Role Of Lcn2 Research Articles

Lipocalin-2: A Nurturer of Tumor Progression and a Novel Candidate for Targeted Cancer Therapy

Within the tumor microenvironment (TME) exists a complex signaling network between cancer cells and stromal cells, which determines the fate of tumor progression. Hence, interfering with this signaling network forms the basis for cancer therapy. Yet, many types of cancer, in particular, solid tumors, are refractory to the currently used treatments, so there is an urgent need for novel molecular targets that could improve current anti-cancer therapeutic strategies. Lipocalin-2 (Lcn-2), a secreted siderophore-binding glycoprotein that regulates iron homeostasis, is highly upregulated in various cancer types. Due to its pleiotropic role in the crosstalk between cancer cells and stromal cells, favoring tumor progression, it could be considered as a novel biomarker for prognostic and therapeutic purposes. However, the exact signaling route by which Lcn-2 promotes tumorigenesis remains unknown, and Lcn-2-targeting moieties are largely uninvestigated. This review will (i) provide an overview on the role of Lcn-2 in orchestrating the TME at the level of iron homeostasis, macrophage polarization, extracellular matrix remodeling, and cell migration and survival, and (ii) discuss the potential of Lcn-2 as a promising novel drug target that should be pursued in future translational research.

The role of LCN2 and LCN2-MMP9 in spondylitis radiographic development: gender and HLA-B27 status differences

BackgroundMale HLA-B27-positive radiographic-axial spondyloarthritis (r-axSpA) patients are prone to have severe spinal radiographic progression, but the underlying mechanisms are unclear. We recently showed that persistently elevated Lipocalin 2 (LCN2; L) reflects sacroiliac joint (SIJ) inflammation. LCN2 binds to MMP9. Concomitant elevation of L and LCN2-MMP9 (LM) was detected in many inflammatory diseases. We asked whether L and LM play similar roles in r-axSpA pathogenesis.MethodsWe analyzed 190 axSpA patients (123 radiographic and 67 non-radiographic axSpA) who had no detectable circulating Oncostatin M, to avoid complications due to cross-talk between pathways. L and LM levels from a single blood sample of each patient were measured and were correlated with MRI and modified stoke AS (mSASS) scoring. Association of elevated L (L+) or concurrent L+ and elevated LM (LM+) patterns with B27 status and gender were assessed.ResultsIn L+LM+ axSpA patients, both L and LM levels correlated with MRI SPARCC SIJ scores, but only LM levels correlated with MRI Berlin Spine Scores, suggesting LM is a biomarker for both SIJ and spinal inflammation. Among patients with minimal spinal ankylosis (mSASSS < 10), 65% of male r-axSpA patients are L+LM+, while 30% and 64% of female patients are L+LM+ and L+, respectively, supporting the role of LM with disease progression. In B27+ L+LM+ male patients, both L and LM (but not CRP) levels correlate with mSASSS. B27 positivity and maleness have additive effects on spondylitis progression, suggesting concurrent high L and LM elevations are associated with B27+ male patients having more significant radiographic damage. L+ B27-negative male patients or L+ female patients are more likely to have milder disease.ConclusionL and LM are informative biomarkers for SIJ and spinal inflammation, as well as for ankylosing development in r-axSpA patients. Distinctive L+LM+ or L+ patterns not only could distinguish clinically aggressive vs milder course of disease, respectively, but also provide an explanation for B27-positive male patients being the most susceptible to severe ankylosis.

309-OR: Lipocalin 2 Deficiency Impairs Mitochondrial Dynamics and Phospholipid Metabolism in Brown Adipocytes

Impaired mitochondrial bioenergetics in brown adipocytes links to the disrupted energy metabolism and thermogenesis in obesity. Phospholipids, particularly cardiolipin and phosphatidic acid (PA) are critical regulators of mitochondrial membrane architecture and dynamics. Mitochondria-associated endoplasmic reticulum membranes (MAMs) serve as the platform for phospholipid biosynthesis. MAM dysfunction disturbs phospholipid metabolism, thereby disrupting mitochondrial function. However, little is known about the participating factors that regulate MAM phospholipid metabolism. We have previously characterized the role of lipocalin 2 (Lcn2) in thermogenesis and mitochondrial function in adipocytes. Herein, we found that Lcn2 was located at MAMs and had a specific PA binding capability. The mitochondrial fusion-fission process induced by the fasting-refeeding cycle was impaired in Lcn2 knockout (KO) brown adipocytes. We also observed that the levels of phosphorylated Drp1 (Ser616) and Opa1 were decreased in brown adipose tissue (BAT) of Lcn2 KO mice compared to WT controls upon acute cold stimulation, whereas Lcn2 overexpression in BAT led to an increase in Drp1 phosphorylation and Opa1 level. Moreover, Lcn2 deficiency significantly enhanced the cold-stimulated accumulation of Opa1 as well as MAM markers SigmaR1, Ip3R1, and Facl4 at MAMs in BAT, suggesting impaired MAM function. CL316,243, a β3-adrenergic receptor agonist has a cold mimic effect on mitochondrial bioenergetics. In brown adipocytes, Lcn2 deficiency significantly attenuated the CL316,243 induction of Drp1 phosphorylation. Intriguingly, lipidomic analysis of BAT mitochondria isolated from mice treated with CL316,243 for 14 days showed that Lcn2 deficiency blunted CL316,243-induced cardiolipin biosynthesis and altered phospholipid metabolism. Together, we discovered a role of Lcn2 as a novel PA transfer protein in mitochondrial bioenergetics via regulating phospholipid metabolism. Disclosure H.Su: None. H.Guo: None. X.Qiu: None. T.Lin: None. X.Chen: None. Funding This work was supported by NIDDK Grant (RDK123042) awarded to Xiaoli Chen.

Role of lipocalin-2 in surgery-induced cognitive decline in mice: a signal from neuron to microglia

BackgroundPerioperative neurocognitive disorders (PNDs) are common complications observed among surgical patients. Accumulating evidence suggests that neuroinflammation is one of the major contributors to the development of PNDs, but the underlying mechanisms remain unclear.MethodsqPCR and ELISA analysis were used for detecting LCN2 and cytokine levels. cx3cr1CreER/−:: R26iDTR/− crossed mouse line was used for microglia depletion; intracranial injection of recombinant LCN2 (rLCN2) and adeno-associated viruses (AAV)-mediated shRNA silencing approaches were used for gain and loss of function, respectively. Combing with in vitro microglia cell culture, we have studied the role of LCN2 in surgery-induced cognitive decline in mice.ResultsWe revealed that Lcn2 mRNA and protein levels were greatly increased in mouse hippocampal neurons after surgery. This surgery-induced elevation of LCN2 was independent of the presence of microglia. Gain of function by intracranial injection of rLCN2 protein into hippocampus disrupted fear memory in naive mice without surgery. Conversely, silencing LCN2 in hippocampus by AAV-shRNA protected mice from surgery-induced microglia morphological changes, neuroinflammation and cognitive decline. In vitro, application of rLCN2 protein induced the expression of several pro-inflammatory cytokines in both BV-2 and primary microglia culture.ConclusionsThese data suggest LCN2 acts as a signal from neuron to induce proinflammatory microglia, which contributes to surgery-induced neuroinflammation and cognitive decline in mice.

Cardiometabolic associations of circulating Lipocalin-2 in adults with varying degrees of adiposity and insulin resistance

Lipocalin (Lcn2) acts as a mediator of several inflammatory processes. However, evidence on the role of Lcn2 in regulating adiposity and type 2 diabetes mellitus (T2DM) is scarce. Therefore, our study aimed to evaluate the associations between Lcn2 and cardiometabolic parameters among Arab adults with varying degrees of adiposity and insulin resistance. A total of 819 adult Saudi participants (307 males and 512 females) with and without T2DM were included. Anthropometrics, metabolic profile and circulating Lcn2 were assessed. Circulating Lcn2 was significantly highest in the obese group independent of age and sex (p = 0.004), but this significance was lost after stratification to T2DM status. Comparison within age groups revealed that among T2DM participants aged 60–70 years, Lcn2 levels were significantly lower than their younger counterparts in both non-obese and obese groups (unadjusted p = 0.04 and < 0.001, respectively). Lcn2 was inversely associated with diastolic blood pressure (R = −0.25, p = 0.01) and triglycerides (R = 0.23; p = 0.04) as well as positively associated with adiponectin (R = 0.27; p = 0.005) among non-obese, non-T2DM participants. In the T2DM/obese group (N = 328), Lcn2 was inversely associated with age (R = −0.15; p = 0.008) and positively associated with HDL-cholesterol (R = 0.12; p = 0.04) and adiponectin (R = 0.17; p = 0.002). Our study suggests that circulating Lcn2 maybe protective against obesity and T2DM, but prospective studies are needed to confirm findings.

Lipocalin 2 Deficiency Restrains Aging-Related Reshaping of Gut Microbiota Structure and Metabolism.

Gut microbiota modulate age-associated changes in metabolism, innate immune responses, and cognitive function. However, the involvement of host factors in the regulation of age-dependent gut microbial structure and intestinal inflammation is largely unknown. Lipocalin 2 (Lcn2) has previously been identified as an adipocytokine and characterized as an important regulator of diet-induced obesity and inflammation. Previous studies have shown that Lcn2 plays a role in high fat diet-induced reshaping of gut microbiota and intestinal inflammation. However, the role of Lcn2 in the regulation of aging-related reshaping of gut microbiota is unclear. Herein, we demonstrate that fecal levels of Lcn2 are reduced during aging. Age reshaped gut microbiota composition in wild-type (WT) mice. Interestingly, Lcn2 deficiency diminished this effect of aging in Lcn2 knockout (LKO) mice, leading to decreased bacterial diversity and increased Firmicutes to Bacteroidetes (F to B) ratio. Specifically, we identified 16 bacteria at the family level that were differentially abundant between WT and LKO mice at old age. Several health-promoting bacteria, including SCFA-producing bacteria, were significantly less prevalent in old LKO mice compared to WT mice, indicating that Lcn2 deficiency shifts the aging-related gut microbial community towards an unhealthy population and lowers microbial butyrate production. Our results provide a line of evidence that Lcn2 plays a role in the control of aging-related reshaping of gut microbiota composition and metabolites.

The interaction between lipocalin 2 and dipyridine ketone hydrazone dithiocarbamte may influence respective function in proliferation and metastasis-related gene expressions in HepG2 cell.

LCN2 (Lipocalins) was first identified as iron transporter through associating with its siderophores and also involved in many cancer metastases, but its function is still paradoxical. We questioned that whether LCN2 might also associate exogenous iron chelator as does in inherent way and the association may influence their respective function. To address this issue, we investigated the effect of LCN2 on action of DpdtC (2,2'-dipyridine ketone hydrazone dithiocarbamte), an iron chelator in proliferation and metastasis-related gene expression. The results showed that exogenous LCN2 and DpdtC could inhibit growth of HepG2 cells, while the combination treatment enhanced their inhibitory effect both in proliferation and colony formation. This encouraged us to investigate the effect of the interaction on metastasis-related gene expression. The results revealed that both LCN2 and DpdtC impaired the wound healing of HepG2, but the inhibitory effect of DpdtC was significantly enhanced upon association with LCN2. Undergoing epithelium-mesenchymal transition (EMT) is a crucial step for cancer metastasis, LCN2 and DpdtC had opposite effects on EMT markers, the binding of DpdtC to LCN2 significantly weakened the regulation of it (or its iron chelate) on EMT markers. To insight into the interaction between LCN2 and DpdtC-iron, fluorescence titration and molecular docking were performed to obtain the association constant (~ 104M-1) and thermodynamic parameters (ΔG = -26.10kJ/mol). Importantly this study provided evidence that siderophores-loading state of LCN2 may influence its function, which be helpful for understanding the contradictory role of LCN2 in the metastasis of cancer.

An Integrative Pan-Cancer Analysis Revealing LCN2 as an Oncogenic Immune Protein in Tumor Microenvironment.

BackgroundLipocalin 2 (LCN2), an innate immune protein, plays a pivotal role in promoting sterile inflammation by regulating immune responses. However, the role of LCN2 in diverse cancers remains poorly defined. This research aimed to investigate the correlation between LCN2 expression and immunity and visualize its prognostic landscape in pan-cancer.MethodsRaw data in regard to LCN2 expression in cancer patients were acquired from TCGA and GTEx databases. Besides, we investigated the genomic alterations, expression pattern, and survival analysis of LCN2 in pan-cancer across numerous databases, including cBioPortal and GEPIA database. The correlation between LCN2 expression and tumor immune infiltration was explored via TIMER, and we utilized CIBERSORT and ESTIMATE computational methods to assess the proportion of tumor-infiltrating immune cells (TIICs) and the amount of stromal and immune components from TCGA database. Protein–Protein Interaction analysis was performed in GeneMANIA database, and gene functional enrichment was performed by Gene Set Enrichment Analysis (GSEA).ResultsOn balance, tumor tissue had a higher LCN2 expression level compared with that in normal tissue. Elevated expression of LCN2 was related to poor clinical regimen with OS and RFS. There were significant positive correlations between LCN2 expression and TIICs, including CD8+ T cells, CD4+ T cells, B cells, neutrophils, macrophages, and dendritic cells. Moreover, markers of TIICs exhibited different LCN2-related immune infiltration patterns. GSEA analysis showed that the expression of LCN2 was related to retinol metabolism, drug metabolism cytochrome P450 and metabolism of xenobiotics by cytochrome P450.ConclusionsThese findings suggested that LCN2 might serve as a biomarker for immune infiltration and poor prognosis in cancers, shedding new light on therapeutics of cancers.

Identification of gut microbiota and microbial metabolites regulated by an antimicrobial peptide lipocalin 2 in high fat diet-induced obesity.

Lipocalin 2 (Lcn2), as an anti-microbial peptide is expressed in intestine, and the upregulation of intestinal Lcn2 has been linked to inflammatory bowel disease. However, the role of Lcn2 in shaping gut microbiota during diet-induced obesity (DIO) remains unknown. We found that short-term high fat diet (HFD) feeding strongly stimulates intestinal Lcn2 expression and secretion into the gut lumen. As the HFD feeding prolongs, fecal Lcn2 levels turn to decrease. Lcn2 deficiency accelerates the development of HFD-induced intestinal inflammation and microbiota dysbiosis. Moreover, Lcn2 deficiency leads to the remodeling of microbiota-derived metabolome, including decreased production of short-chain fatty acids (SCFAs) and SCFA-producing microbes. Most importantly, we have identified Lcn2-targeted bacteria and microbiota-derived metabolites that potentially play roles in DIO and metabolic dysregulation. Correlation analyses suggest that Lcn2-targeted Dubosiella and Angelakisella have a novel role in regulating SCFAs production and obesity. Our results provide a novel mechanism involving Lcn2 as an anti-microbial host factor in the control of gut microbiota symbiosis during DIO.

Abstract P231: Macrophage Neutrophil Gelatinase-associated Lipocalin Has A Critical Role In Aldosterone-induced Renal Fibrosis Via The Ccl5-il4 Pathway.

Introduction: Neutrophil Gelatinase-Associated Lipocalin (NGAL) (or lipocalin 2) is a novel mineralocorticoid biotarget in the cardiovascular system. NGAL is also described as an acute renal lesion biomarker and NGAL serum concentration is associated with the severity of renal damages patients with a chronic kidney disease (CKD). Lipocalin2 (Lcn2) gene invalidation in a CKD mouse model protects from proteinuria and renal lesions. Objective: We hypothesized that the NGAL from macrophages promotes expression of chemoattractant molecules involved in renal lesions induced by mineralocorticoid excess. Methods: The role of Lcn2 was analyzed using full Lcn2 knock out mice (NGAL KO) challenged with uni-Nephrectomy, Aldosterone 200μg/kg/day, Salt 1% (NAS model) during 6 weeks. Assessment of CCL5/IL4 in kidney fibrosis were studied using maraviroc administration (50mg/kg/d in chow diet) or by injections of anti-IL4 antibody (600μg/week). Results: NAS induced a significant increase in the expression (relative values, mean±SEM, compared to 1 in the control samples, p&amp;lt0.05) of extracellular matrix proteins such as collagen I (2.35±0.33), α-SMA (2.04±0.44) and fibronectin (3.38±0.42) in the kidney of WT mice associated with interstitial kidney fibrosis (6.49±0.70). These modifications were fully prevented by NGAL deletion. Expression of macrophages markers F4/80 , CD80 and CD86 was increased (5.11±0.46, 4.84±0.19 and 5.22±0.45 respectively) in WT NAS mice and partly prevented in NGAL KO mice. Macrophages isolated from NGAL KO or WT mice were co-treated with aldosterone (10 -8 M) and NaCl (40mM). In WT macrophages, expression of Lcn2 (2.81±0.30) and the CCL5 chemokine (2.48±0.32) was increased. The increase of CCL5 was prevented in NGAL KO macrophages. Such as total deletion of NGAL, CCL5 receptor blockade improved renal fibrosis and high level of Th2 CD4 + cell markers induced by NAS. Neutralization of IL4 Th2 cytokine in NAS mice injected with anti-IL4 antibody blunted kidney fibrosis and overexpression of profibrotic proteins such as collagen I, α-SMA and fibronectin. Conclusion: NGAL produced by macrophages plays a critical role in renal interstitial fibrosis through CCL5/IL4 pathways in mice exposed to mineralocorticoid excess.

Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function.

Lipocalin 2 (Lcn2) is an essential component of the antimicrobial innate immune system. It attenuates bacterial growth by binding and sequestering the iron-scavenging siderophores to prevent bacterial iron acquisition. Whereas, the ability of Lcn2 to sequester iron is well-described, the role of Lcn2 in regulating immune cells during bacterial infection remains unclear. In this study, we showed that upon infection with Escherichia coli (O157:H7), Lcn2-deficient (Lcn2−/−) mice carried more bacteria in blood and liver, and the acute-phase sera lost their antibacterial activity in vitro. Neutrophils from Lcn2−/− mice were defective in homeostasis and morphological development. E. coli O157:H7 infection of Lcn2−/− mice resulted in a reduced neutrophil migration capacity, with 30% reduction of extravasated neutrophils, and impaired chemotaxis, as shown by a reduction in the secretion of chemoattractants, such as tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2, which are instrumental in eliciting a neutrophil response. We also found that some secreted cytokines [interleukin (IL)-6, IL-1β, and TNF-α] were decreased. Transcripts of inflammatory cytokines (IL-6, IL-1β, TNF-α, and IL-10), chemokines (MIP-2 and MCP-1), and iNOS production were all strongly repressed in Lcn2−/− macrophages. Furthermore, Lcn2 could induce the production of chemokines and promote the migration and phagocytosis of macrophages. Thus, Lcn2 deficiency could impair the migration and chemotaxis ability of neutrophils and disturb the normal secretion of inflammatory cytokines of macrophages. Therefore, the heightened sensitivity of Lcn2−/− mice to E. coli O157:H7 is not only due to the antibacterial function of Lcn2 but also a consequence of impaired functions of immune cells, including neutrophils and macrophages.

Abstract 010: Neutrophil Gelatinase Associated Lipocalin From Immune Cells is Involved in Renal Damages Induced by Mineralocorticoid Excess

Introduction: Neutrophil Gelatinase-Associated Lipocalin (NGAL) (or lipocalin 2) is a novel mineralocorticoid biotarget in the cardiovascular system. NGAL is also described as an acute renal lesion biomarker and NGAL serum concentration is associated with the severity of renal damages patients with a chronic kidney disease (CKD). Lipocalin2 (Lcn2) gene invalidation in a CKD mouse model protects from proteinuria and renal lesions. Objective: Since the immune system is involved in renal inflammation and fibrosis process, we hypothesized that the production of NGAL/Lcn2 by immune cells plays an important role in the deleterious mineralocorticoid effects in the kidney. Methods: The role of Lcn2 produced by immune cells was analyzed using full Lcn2 knock out mice (Full KO) and mice depleted for Lcn2 in their immune cells by bone marrow transplantation (BMT lcn2 KO). These mice were challenged with uni-Nephrectomy, Aldosterone 200μg/kg/day, Salt 1% (NAS model) during 6 weeks. Results: NAS induced a significant increase in the expression (relative values, mean±SEM, compared to 1 in the control samples, p&lt;0.05, n=6-9) of proinflammatory proteins such as MCP-1 (5.00±0.26), TNF-α (8.58±0.31), OPN (1.60±0.07), RANTES (3.98±0.20) and extracellular matrix proteins such as collagen IV (1.94±0.10), α-SMA (2.04±0.13) and fibronectin (3.38±0.20) in the kidney of WT mice. This effect was blunted in both KO mouse models (Full KO and BMT KO). Kidney fibrosis was induced by NAS (relative values, mean±SEM, compared to 1 in the control samples, p&lt;0.05, n= 6-9) (5.98±0.19). this increase was fully prevented in Full KO NAS mice and partly corrected in BMT KO NAS mice. Macrophages isolated from Full Lcn2 KO or WT mice were co-treated with aldosterone (10 -8 M) and NaCl (40mM). In WT macrophages, mRNA expression (relative values, mean±SEM, compared to 1 in the control samples, p&lt;0.05, n=6) of Lcn2 (22.06±1.36) as well as a the RANTES/CCl5 chemokine (10.88±0.88) is increased. The increase of RANTES/CCl5 was fully blunted in Lcn2 KO macrophages. Conclusion: NGAL produced by immune cells plays a critical role in renal interstitial fibrosis and inflammation induced by mineralocorticoid excess. The role of RANTES/CCl5 in the renal NGAL-mineralocorticoid pathways is currently analyzed.

Abstract 4952: Host lipocalin 2 protects against Kras mutant lung cancer development by maintaining an anti-tumor immune contexture

Abstract Relative to other lung adenocarcinomas (LUADs), Kras mutant LUAD displays dismal prognosis warranting the need for new strategies for early treatment of this lung cancer subtype. Limiting these advances is our poor understanding of events that drive Kras mutant LUAD development. In efforts to fill this void, we recently found that mice with knockout of G-protein coupled receptor 5A (Gprc5a-/-) and after tobacco carcinogen exposure (nicotine-specific nitrosamine ketone/NNK) developed LUADs harboring driver somatic Kras mutations. To understand early events preceding the onset of these Kras mutant LUADs, we performed RNA-sequencing (RNA-Seq) of normal airways in Gprc5a-/- mice and wild type (WT) littermates prior to NNK exposure. We found markedly elevated expression of the antimicrobial lipocalin 2 (Lcn2) gene in normal airways of Gprc5a-/- mice. Also, analysis of publicly available human datasets revealed elevated expression of LCN2 in human LUADs relative to normal lung tissues particularly in KRAS mutant tumors. We then sought to probe the role of Lcn2 in the pathogenesis of Kras mutant LUAD. Mice with knockout of host Lcn2 (Gprc5a-/-/Lcn2-/-) showed increased lung tumor development compared to Gprc5a-/- littermates. RNA-Seq of whole lungs at baseline and at seven months post-NNK (n = 9-10 mice each group) pinpointed differential expression profiles that denoted decreased anti-tumor immunity in Gprc5a-/-/Lcn2-/- mice. Computational estimation of immune infiltrates demonstrated increased levels of neutrophils post-NNK in Gprc5a-/-/Lcn2-/- mice but not in Gprc5a-/-littermates. By flow cytometry analysis of lung tissues, we found elevated numbers of various lymphoid and myeloid immune cells in both groups following NNK exposure and increased neutrophil counts in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- littermates. Histopathological assessment also revealed increased numbers of pro-inflammatory lesions in mice lacking Lcn2. Gprc5a-/-/Lcn2-/- mice at three months post-NNK were also found to exhibit increased production of IL-17A by lung-resident CD4+ and CD8+ T cells relative to Gprc5a-/- littermates concomitant with decreased expression of the anti-tumor Th1/cytolytic immune markers Ifnγ and Gzmb. Preliminary analysis also showed elevated expression of the major immune checkpoint PD-L1 on alveolar macrophages in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- mice with WT Lcn2. Lastly, we preliminarily found, by bacterial 16S rRNA sequencing of temporally collected fecal samples, globally altered microbiome profiles in mice lacking Lcn2. Our findings underscore novel cues in lung oncogenesis and suggest that Lcn2 decreases anti-tumor immunity, perturbs the host microbiome and promotes development of Kras mutant LUAD thereby offering new venues for early treatment of this fatal malignancy. Citation Format: Maya Hassane, Warapen Treekitkarnmongkol, Tina L. McDowell, Smruthy Sivakumar, Wenhua Lang, Joshua K. Ochieng, Sayuri Nunomura-Nakamura, Casey Finnicum, Christel Davis, Gareth E. Davies, Junya Fukuoka, Tina Cascone, Florencia McAllister, Ignacio I. Wistuba, Erik Ehli, Seyed J. Moghaddam, Paul Scheet, Junya Fujimoto, Humam Kadara. Host lipocalin 2 protects against Kras mutant lung cancer development by maintaining an anti-tumor immune contexture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4952.

2024-P: Protective Role of Lipocalin 2 in High-Fat Diet-Induced Gut Microbiota Dysbiosis

Diet is the most important factor that shapes diversity in gut microbiome. However, what host factors are involved in dietary regulation of gut microbiota remains largely unknown. Lipocalin 2 (Lcn2) is expressed in intestine, and its expression is upregulated during acute intestinal inflammation. Herein, we investigated the role of Lcn2 in shaping gut microbiota during high-fat diet (HFD)-induced obesity. First, we showed that 12 weeks of HFD feeding led to a significant increase in Lcn2 protein levels in ileum and colon, but a decrease in Lcn2 levels in feces of C57BL/6J mice. This suggests that Lcn2 release into the gut lumen decreases following long-term HFD feeding. Second, we revealed a time course of HFD-induced changes in gut microbiota. Feces were collected from wild type (WT) and Lcn2 knockout (LKO) mice at 2, 4, 8 and 16 weeks of HFD feeding for DNA extraction and 16S sequencing. Compared to WT mice, LKO mice displayed a trend towards lower bacterial diversity (p=0.078) at 4 weeks but not 16 weeks of HFD feeding, suggesting that Lcn2 may have a protective role against gut microbiota dysbiosis during short-term HFD consumption. More interestingly, species richness (the number of bacterial species) was increased at 4 and 8 weeks of HFD feeding and then declined 16 weeks after HFD feeding in WT mice; this time-dependent change in the richness was not observed in LKO mice. Principal coordinate analysis indicates a difference in gut microbiota composition between WT and LKO mice when all time points were considered for the analysis. The heatmap shows that the abundance of short-chain fatty acid-producing bacteria such as Lachnospiraceae and Muribaculaceae was significantly decreased in HFD-fed LKO mice. Lastly, upon 16 weeks of HFD feeding, LKO mice had increased levels of STAT3 and NFκB phosphorylation in colon compared to WT mice, suggesting increased intestinal inflammation. We conclude that Lcn2 plays a protective role in HFD-induced intestinal inflammation and gut microbiota dysbiosis. Disclosure X. Qiu: None. M. Macchietto: None. S. Shen: None. X. Chen: None.

Lipocalin 2 contributes to brain iron dysregulation but does not affect cognition, plaque load, and glial activation in the J20 Alzheimer mouse model

BackgroundLipocalin 2 (Lcn2) is an acute-phase protein implicated in multiple neurodegenerative conditions. Interestingly, both neuroprotective and neurodegenerative effects have been described for Lcn2. Increased Lcn2 levels were found in human post-mortem Alzheimer (AD) brain tissue, and in vitro studies indicated that Lcn2 aggravates amyloid-β-induced toxicity. However, the role of Lcn2 has not been studied in an in vivo AD model. Therefore, in the current study, the effects of Lcn2 were studied in the J20 mouse model of AD.MethodsJ20 mice and Lcn2-deficient J20 (J20xLcn2 KO) mice were compared at the behavioral and neuropathological level.ResultsJ20xLcn2 KO and J20 mice presented equally strong AD-like behavioral changes, cognitive impairment, plaque load, and glial activation. Interestingly, hippocampal iron accumulation was significantly decreased in J20xLcn2 KO mice as compared to J20 mice.ConclusionsLcn2 contributes to AD-like brain iron dysregulation, and future research should further explore the importance of Lcn2 in AD.

Lipocalin2 mediates anti-inflammatory functions through the inhibition of STAT3 and activation of STAT5 signaling in bone marrow derived macrophages

Abstract Lipocalin2 (Lcn2), an innate immune protein, is known to be significantly up-regulated during inflammatory disorders including arthritis, however, its molecular mechanisms remain to be elucidated. We have shown that Lcn2-knockout (Lcn2KO) mice developed more severe serum-induced arthritis (STA) compared to wild-type (WT) mice with significantly reduced neutrophil infiltration but considerably more macrophage migration. Therefore, we have investigated the possible role of Lcn2 in regulating macrophages. Bone marrow cells were isolated from Lcn2KO and WT mice and polarized to classical or inflammatory (M1) and alternate or anti-inflammatory (M2) macrophages. The polarization of M1 (iNOS) and M2 (arginase-1) phenotypes was confirmed by Western blot analysis. We did not observe a difference in polarization of Lcn2KO and WT macrophages suggesting that Lcn2 may not alter the polarization of macrophages. However, anti-inflammatory cytokines such as TGF-β1 and IL-10 were significantly reduced in either LPS or IC-stimulated M2 phenotypes from Lcn2KO mice compared to WT mice. In agreement, we observed systemic elevated levels of pro-inflammatory cytokines in Lcn2KO compared to WT mice during STA. Further, we’ve demonstrated that Lcn2 deficient M1 macrophages displayed increased STAT3 activation compared to WT cells. In addition, WT M2 phenotype exhibited elevated STAT5 activation. Taken together, these results suggest that Lcn2 may promote the down-regulation of pro-inflammatory cytokines and up-regulate anti-inflammatory cytokines possibly through STAT3 and STAT5 signaling pathways as a potential negative feedback loop in order to limit the extent of inflammation during autoimmune arthritis conditions.

Expression patterns and changes of the LCN2 gene in the testes of induced cryptorchidism and busulfan-treated mice

ABSTRACTLipocalin-2 (LCN2) was known to play various roles in different type cells; however, little was known about the effect of LCN2 in male fertility. In this study, we aimed to explore the expression pattern of LCN2 with increasing age in mice, and to obtain insight into the role of LCN2 in mice testes by induced cryptorchidism and busulfan-treated infertility. In situ hybridization showed that LCN2 was localized primarily in Leydig cells, but was absent in Sertoli and germ cells. Its expression in testes exhibited an age-related increase from day 1 to 8 months, then reduced by the twelth month. The mRNA and protein levels of LCN2 in the testes of both infertile models increased as measured by real-time PCR and western blotting, respectively. LCN2 mRNA and protein levels were higher (p<0.05) in busulfan treated mice than that of cryptorchidism. These observations have shown that LCN2 is developmentally regulated and highly expressed in the Leydig cells of mouse testes.

Abstract A22: The effects of Lipocalin-2 in the early stages of colorectal cancer metastasis

Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. When diagnosed at an early, localized stage, the five year survival rate is approximately 90%; however, when patients are diagnosed at the metastatic stage, this drops to 12%. Metastasis, the spread of cancer cells from the primary tumor to a distant organ, is the primary cause of death in cancer patients. In CRC, metastasis is typically found in the liver, asymptomatic, and often detected at the final stage when therapeutic options are few. The genetic and cellular mechanisms driving the early stages of metastasis are still poorly understood. Recent studies have shown that prior to the arrival of cancer cells at the secondary organ, molecular signals from the tumor direct the recruitment of bone marrow derived cells to create a pre-metastatic niche where cancer cells can attach and develop into a metastatic lesion. Identifying and understanding these molecules can lead to the development of methods for early diagnosis or targets for intervention. Using an orthotopic model of CRC liver metastasis, we performed microarray analyses of the liver microenvironment in tumor bearing mice before and after the arrival of metastatic cells. We found that Lipocalin-2 (LCN2, 24p3, NGAL) was highly expressed in the liver and in circulation of mice bearing highly metastatic cells. LCN2 is a 25 kD secreted glycoprotein that binds small lipophilic molecules. Its expression is upregulated in a number of primary tumors of epithelial origin, but its role in metastasis remains unclear. It has contrasting roles in metastasis in breast cancer models and has been associated with poor prognosis in CRC patients; however its role in the tumor microenvironment has not been studied. Our studies thus far show that overexpression of Lcn2 in mouse colon cancer cells either had little impact on tumor growth and invasiveness, or it enhanced invasion in the presence of macrophages; however, it resulted in an overall reduction of circulating iron levels in sera from tumor bearing mice. In-vitro, iron also seems to have an effect in conjunction with Lcn2 on the invasiveness of the tumor cells. The results from this study will allow us to better describe the role of LCN2 in tumor and stromal cells in the early stages of liver metastasis. Citation Format: Daniel Hughes, Celestia Davis, Yu Zhang, John Bonaparte, Maria Pena. The effects of Lipocalin-2 in the early stages of colorectal cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A22.

Expression and localisation of brain-type organic cation transporter (BOCT/24p3R/LCN2R) in the normal rat hippocampus and after kainate-induced excitotoxicity

The iron siderophore binding protein lipocalin 2 (LCN2, also known as 24p3, NGAL and siderocalin) may be involved in iron homeostasis, but to date, little is known about expression of its putative receptor, brain-type organic cation transporter (BOCT, also known as BOCT1, 24p3R, NGALR and LCN2R), in the brain during neurodegeneration. The present study was carried out to elucidate the expression of LCN2 and BOCT in hippocampus after excitotoxicity induced by the glutamate analog, kainate (KA) and a possible role of LCN2 in neuronal injury. As reported previously, a rapid and sustained induction in expression of LCN2 was found in the hippocampus after intracerebroventicular injection of KA. BOCT was expressed in neurons of the saline-injected control hippocampus, and immunolabel for BOCT protein was preserved in pyramidal neurons of CA1 at 1 day post-KA injection, likely due to the delayed onset of neurodegeneration after KA injection. At 3 days and 2 weeks after KA injections, loss of immunolabel was observed due to degenerated neurons, although remaining neurons continued to express BOCT, and induction of BOCT was found in OX-42 positive microglia. This resulted in an overall decrease in BOCT mRNA and protein expression after KA treatment. Increased expression of the pro-apoptotic marker, Bim, was found in both neurons and microglia after KA injection, but TUNEL staining indicating apoptosis was found primarily in Bim-expressing neurons, but not microglia. Interaction between LCN2 and BOCT was found by DuoLink assay in cultured hippocampal neurons. Apo-LCN2 without iron caused no significant differences in neuronal Bim expression or cell survival, whereas holo-LCN2 consisting of LCN2:iron:enterochelin complex increased Bim mRNA expression and decreased neuronal survival. Together, results suggest that LCN2 and BOCT may have a role in neuronal injury.

The Roles of Lipocalin-2 in Small-for-Size Fatty Liver Graft Injury

To investigate the roles and underlying mechanism of an inflammatory mediator-lipocalin-2 (Lcn2) in small-for-size fatty graft liver injury. Understanding of the distinct mechanism regulating small-for-size fatty liver graft injury will be crucial to prevent marginal graft failure during living donor liver transplantation (LDLT). The roles of Lcn2 in small fatty graft injury were investigated in orthotopic liver transplantation model rats, human LDLT samples, an in vitro simulated ischemia-reperfusion (IR) model, and a hepatic ischemic reperfusion plus major hepatectomy (IR + H) model in mice. Our result showed that Lcn2 was significantly upregulated together with elevation of chemokine (C-X-C motif) ligand 10 (CXCL10) and activation/infiltration of intragraft macrophages after liver transplantation using small-for-size fatty liver graft compared with that of using small-for-size normal liver graft. Intragraft and plasma levels of Lcn2 were intensified in patients who underwent transplantation with small-for-size fatty graft after LDLT. Lcn2 and CXCL10 were expressed higher in fatty hepatocytes after the simulated IR injury compared with normal hepatocytes. Overexpression of Lcn2 significantly deteriorated IR + H-induced hepatic injury in correlation with upregulation of CXCL10 and augmentation of infiltrated macrophages. On the contrary, hepatic injury of small fatty liver remnant after IR + H operation was attenuated in the Lcn-2 mice because of suppression of CXCL10 expression and diminishment of macrophage infiltration. Lcn2 is an important regulator in small-for-size fatty liver graft injury and targeting Lcn2 may be feasible for preventing marginal graft failure in LDLT.