Targeted proteomic methods can accelerate the verification of multiple tumor marker candidates in large series of patient samples. We utilized the targeted approach known as selected/multiple reaction monitoring (S/MRM) to verify potential protein markers of colorectal adenoma identified by our group in previous transcriptomic and quantitative shotgun proteomic studies of a large cohort of precancerous colorectal lesions. We developed SRM assays to reproducibly detect and quantify 25 (62.5%) of the 40 selected proteins in an independent series of precancerous and cancerous tissue samples (19 adenoma/normal mucosa pairs; 17 adenocarcinoma/normal mucosa pairs). Twenty-three proteins were significantly up-regulated (n = 17) or downregulated (n = 6) in adenomas and/or adenocarcinomas, as compared with normal mucosa (linear fold changes ≥ ±1.3, adjusted p value <0.05). Most changes were observed in both tumor types (up-regulation of ANP32A, ANXA3, SORD, LDHA, LCN2, NCL, S100A11, SERPINB5, CDV3, OLFM4, and REG4; downregulation of ARF6 and PGM5), and a five-protein biomarker signature distinguished neoplastic tissue from normal mucosa with a maximum area under the receiver operating curve greater than 0.83. Other changes were specific for adenomas (PPA1 and PPA2 up-regulation; KCTD12 downregulation) or adenocarcinoma (ANP32B, G6PD, RCN1, and SET up-regulation; downregulated AKR1B1, APEX1, and PPA1). Some changes significantly correlated with a few patient- or tumor-related phenotypes. Twenty-two (96%) of the 23 proteins have a potential to be released from the tumors into the bloodstream, and their detectability in plasma has been previously reported. The proteins identified in this study expand the pool of biomarker candidates that can be used to develop a standardized precolonoscopy blood test for the early detection of colorectal tumors.

Triple-negative breast cancer (TNBC), which has the highest mortality rate of all breast cancer, is in urgent need of a therapeutic that hinders the spread and growth of cancer cells. CRISPR genome editing holds the promise of a potential cure for many genetic diseases, including TNBC; however, its clinical translation is being challenged by the lack of safe and effective nonviral delivery systems for in vivo therapeutic genome editing. Here we report the synthesis and application of a noncationic, deformable, and tumor-targeted nanolipogel system (tNLG) for CRISPR genome editing in TNBC tumors. We have demonstrated that tNLGs mediate a potent CRISPR knockout of Lipocalin 2 (Lcn2), a known breast cancer oncogene, in human TNBC cells in vitro and in vivo. The loss of Lcn2 significantly inhibits the migration and the mesenchymal phenotype of human TNBC cells and subsequently attenuates TNBC aggressiveness. In an orthotopic TNBC model, we have shown that systemically administered tNLGs mediated >81% CRISPR knockout of Lcn2 in TNBC tumor tissues, resulting in significant tumor growth suppression (>77%). Our proof-of-principle results provide experimental evidence that tNLGs can be used as a safe, precise, and effective delivery approach for in vivo CRISPR genome editing in TNBC.

BACKGROUND

We are using ACI and BN rats, which differ markedly in their susceptibility to 17β-estradiol (E2)-induced mammary cancer, to identify genetic variants and environmental factors that determine mammary cancer susceptibility. The objective of this study was to characterize the cellular and molecular responses to E2 in the mammary glands of ACI and BN rats to identify qualitative and quantitative phenotypes that associate with and/or may confer differences in susceptibility to mammary cancer.

METHODS

Female ACI and BN rats were treated with E2 for 1, 3 or 12 weeks. Mammary gland morphology and histology were examined by whole mount and hematoxylin and eosin (H&E) staining. Cell proliferation and epithelial density were evaluated by quantitative immunohistochemistry. Apoptosis was evaluated by quantitative western blotting and flow cytometry. Mammary gland differentiation was examined by immunohistochemistry. Gene expression was evaluated by microarray, qRT-PCR and quantitative western blotting assays. Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy.

RESULTS

The luminal epithelium of ACI rats exhibited a rapid and sustained proliferative response to E2. By contrast, the proliferative response exhibited by the mammary epithelium of BN rats was restrained and transitory. Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM. Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed.

CONCLUSIONS

We propose that these cellular and molecular phenotypes are heritable and may underlie, at least in part, the differences in mammary cancer susceptibility exhibited by ACI and BN rats.

Interleukin 22 (IL-22) is an IL-10-related cytokine produced by T helper 17 (Th17) cells and other immune cells that signals via IL-22 receptor alpha 1 (IL-22Ra1), which is expressed on epithelial tissues, as well as hepatocytes. IL-22 has been shown to have hepatoprotective effects that are mediated by signal transducer and activator of transcription 3 (STAT3) signaling. However, it is unclear whether IL-22 can directly regulate antimicrobial programs in the liver. To test this hypothesis, hepatocyte-specific IL-22Ra1 knockout (Il22Ra1(Hep-/-)) and Stat3 knockout (Stat3(Hep-/-)) mice were generated and subjected to intra-abdominal infection with Klebsiella pneumoniae, which results in liver injury and necrosis. We found that overexpression of IL-22 or therapeutic administration of recombinant IL-22 (rIL-22), given 2 h postinfection, significantly reduced the bacterial burden in both the liver and spleen. The antimicrobial activity of rIL-22 required hepatic Il22Ra1 and Stat3. Serum from rIL-22-treated mice showed potent bacteriostatic activity against K. pneumoniae, which was dependent on lipocalin 2 (LCN2). However, in vivo, rIL-22-induced antimicrobial activity was only partially reduced in LCN2-deficient mice. We found that rIL-22 also induced serum amyloid A2 (SAA2) and that SAA2 had anti-K. pneumoniae bactericidal activity in vitro. These results demonstrate that IL-22, through IL-22Ra1 and STAT3 singling, can induce intrinsic antimicrobial activity in the liver, which is due in part to LCN2 and SAA2. Therefore, IL-22 may be a useful adjunct in treating hepatic and intra-abdominal infections.

The neutrophil gelatinase-associated lipocalin (NGAL, also known as LCN2) and its cellular receptor (LCN2-R, SLC22A17) are involved in many physiological and pathological processes such as cell differentiation, apoptosis, and inflammation. These pleiotropic functions mainly rely on NGAL's siderophore-mediated iron transport properties. However, the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo state to form a fuzzy complex. The relatively weak affinity (≈10 μm) between human LCN2-R-NTD and apoNGAL suggests that the N terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, human LCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.

Cervical carcinoma is the third-most common cause of cancer-related deaths in women worldwide. However, the molecular mechanisms underlying the metastasis of cervical cancer are still unclear. Oligonucleotide microarrays coupled with bioinformatics analysis show that cytoskeletal remodeling and epithelial-to- mesenchymal transition (EMT) are significant pathways in clinical specimens of cervical cancer. In accord with clinical observations demonstrating ectopic expression of lipocalin 2 (LCN2), an oncogenic protein associated with EMT, in malignant tumors, was significantly upregulated in cervical cancer and correlated with lymph node metastasis. Overexpression of LCN2 enhanced tumor cell migration and invasion both in vitro and in vivo. Conversely, knockdown or neutralization of LCN2 reduced tumor cell migration and invasion. LCN2-induced migration was stimulated by activation of the EMT-associated proteins, Snail, Twist, N-cadherin, fibronectin, and MMP-9. Our findings collectively support a potential role of LCN2 in cancer cell invasion through the EMT pathway and suggest that LCN2 could be effectively utilized as a lymph node metastasis marker in cervical cancer.

The control over iron availability is crucial under homeostatic conditions and even more in the case of an infection. This results from diverse properties of iron: first, iron is an important trace element for the host as well as for the pathogen for various cellular and metabolic processes, second, free iron catalyzes Fenton reaction and is therefore producing reactive oxygen species as a part of the host defense machinery, third, iron exhibits important effects on immune cell function and differentiation and fourth almost every immune activation in turn impacts on iron metabolism and spatio-temporal iron distribution. The central importance of iron in the host and microbe interplay and thus for the course of infections led to diverse strategies to restrict iron for invading pathogens. In this review, we focus on how iron restriction to the pathogen is a powerful innate immune defense mechanism of the host called "nutritional immunity". Important proteins in the iron-host-pathogen interplay will be discussed as well as the influence of iron on the efficacy of innate and adaptive immunity. Recently described processes like ferritinophagy and ferroptosis are further covered in respect to their impact on inflammation and infection control and how they impact on our understanding of the interaction of host and pathogen.

Keywords: Calprotectin; Ferritin; Ferritinophagy; Ferroportin; Ferroptosis; HFE; IRP; Iron; Lactoferrin; Lcn2; NO; NRAMP1; Nutritional immunity, Anemia, infection, Heme metabolism.

OBJECTIVE

Microbiota is a major player in the pathogenesis of inflammatory bowel diseases (IBD) and colorectal cancer (CRC). Here, we summarize the key advances achieved in the past 18 months (ending June 2017) toward a better understanding of the role of microbiota in colitis and CRC development.

RESULTS

Accumulating evidence shows the essential role of intestinal barrier function (e.g. mucus, IgA, LCN2, LYPD8) in protecting against bacteria-induced inflammation and tumor development. Numerous signaling pathways (e.g. TLRs and NLRs), metabolites (e.g. indole, bile acids, retinoic acid) and small noncoding RNAs (e.g. miRNA) have been identified as key mediators regulating host-microbe interactions in the intestine. Novel microbial drivers of colitis and tumorigenesis (e.g. Alistipes finegoldii, Atopobium parvalum, Peptostreptococcus anaerobius) have been identified and their disease-promoting activities have been described.

CONCLUSIONS

IBD-associated colorectal cancer results from a complex breakdown of communication between the host and its microbiota, involving barrier function, immune signaling and metabolites.

A very low 5-year survival rate among hepatocellular carcinoma (HCC) patients is mainly due to lack of early stage diagnosis, distant metastasis and high risk of postoperative recurrence. Hence ascertaining novel biomarkers for early diagnosis and patient specific therapeutics is crucial and urgent. Here, we have performed a comprehensive analysis of the expression data of 423 HCC patients (373 tumors and 50 controls) downloaded from The Cancer Genome Atlas (TCGA) followed by pathway enrichment by gene ontology annotations, subtype classification and overall survival analysis. The differential gene expression analysis using non-parametric Wilcoxon test revealed a total of 479 up-regulated and 91 down-regulated genes in HCC compared to controls. The list of top differentially expressed genes mainly consists of tumor/cancer associated genes, such as AFP, THBS4, LCN2, GPC3, NUF2, etc. The genes over-expressed in HCC were mainly associated with cell cycle pathways. In total, 59 kinases associated genes were found over-expressed in HCC, including TTK, MELK, BUB1, NEK2, BUB1B, AURKB, PLK1, CDK1, PKMYT1, PBK, etc. Overall four distinct HCC subtypes were predicted using consensus clustering method. Each subtype was unique in terms of gene expression, pathway enrichment and median survival. Conclusively, this study has exposed a number of interesting genes which can be exploited in future as potential markers of HCC, diagnostic as well as prognostic and subtype classification may guide for improved and specific therapy.

Interleukin (IL)-6 is critical for the febrile response to peripheral immune challenge. However, the mechanism by which IL-6 enables fever is still unknown. To characterise the IL-6-dependent fever generating pathway, we used microarray analysis to identify differentially expressed genes in the brain of lipopolysaccharide (LPS)-treated IL-6 wild-type and knockout mice. Mice lacking IL-6 displayed a two-fold lower expression of the lipocalin-2 gene (lcn2), and this difference was confirmed by real-time reverse transcriptase-polymerase chain reaction. Conversely, the induction of lipocalin-2 protein was observed in brain vascular cells following i.p. administration of recombinant IL-6, suggesting a direct relationship between IL-6 and lipocalin-2. Immunohistochemical analysis also revealed that LPS-induced lipocalin-2 is expressed by brain endothelial cells and is partly co-localised with cyclooxygenase-2 (Cox-2), the rate-limiting enzyme for the production of inflammatory induced prostaglandin E(2) (PGE(2) ), which is the key mediator of fever. The direct role of lipocalin-2 in fever was examined in LPS-challenged lipocalin-2 knockout mice. In both male and female mice, normal fever responses were observed at near-thermoneutral conditions (29-30 °C) but when recorded at normal room temperature (19-20 °C), the body temperature of lipocalin-2 knockout female mice displayed an attenuated fever response compared to their wild-type littermates. This difference was reflected in significantly attenuated mRNA expression of Cox-2 in the brain of lipocalin-2 knockout female mice, but not of male mice, following challenge with peripheral LPS. Our findings suggest that IL-6 influences the expression of lipocalin-2, which in turn may be involved in the control of the formation of Cox-2, and hence central PGE(2) -production. We have thus identified lipocalin-2 as a new factor in the pathway of inflammatory IL-6 signalling. However, the effect of lipocalin-2 on fever is small, being sex-dependent and ambient temperature-specific, and thus lipocalin-2 cannot be considered as a major mediator of the IL-6-dependent fever generating pathway.

Lipocalin-2 (LCN2) belongs to the superfamily of lipocalins and plays critical roles in the control of cellular homeostasis during inflammation and in responses to cellular stress or injury. In the liver, LCN2 triggers protective effects following acute or chronic injury, and its expression is a reliable indicator of liver damage. However, little is known about LCN2's functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis. In this study, we fed wild type (WT) and LCN2-deficient (Lcn2(-/-)) mice a methionine- and choline-deficient (MCD) diet as a nutritional model of non-alcoholic steatohepatitis, and compared intrahepatic lipid accumulation, lipid droplet formation, mitochondrial content, and expression of the Perilipin proteins that regulate cellular lipid metabolism. We found that Lcn2(-/-) mice fed an MCD diet accumulated more lipids in the liver than WT controls, and that the basal expression of the lipid droplet coat protein Perilipin 5 (PLIN5, also known as OXPAT) was significantly reduced in these animals. Similarly, the overexpression of LCN2 and PLIN5 were also found in animals that were fed with a high fat diet. Furthermore, the loss of LCN2 and/or PLIN5 in hepatocytes prevented normal intracellular lipid droplet formation both in vitro and in vivo. Restoration of LCN2 in Lcn2(-/-) primary hepatocytes by either transfection or adenoviral vector infection induced PLIN5 expression and restored proper lipid droplet formation. Our data indicate that LCN2 is a key modulator of hepatic lipid homeostasis that controls the formation of intracellular lipid droplets by regulating PLIN5 expression. LCN2 may therefore represent a novel therapeutic drug target for the treatment of liver diseases associated with elevated fat accumulation and steatosis.

Lipocalin 24p3 plays a direct role in iron transport and regulates the levels of important proteins of the iron metabolism. Iron-loaded 24p3 binds to its specific receptor (24p3R) on the cell surface. Upon binding to its receptor, 24p3 is internalized into the cell, where it releases its bound iron. Iron-free 24p3 can withdraw iron from inside the cell to the outside by a reverse mechanism. We analyzed the role of the murine 24p3 gene Lcn2 (alias 24p3) as a target of the Wnt pathway. In cells with activated Wnt pathway, the levels of 24p3 protein and RNA were decreased. The withdrawal of iron led to 24p3 downregulation, and iron addition to iron-deprived cells induced 24p3 expression. Despite its strong inhibitory effect on 24p3 expression, Wnt pathway activation had no effect on the intracellular iron level. In cells with nonactivated Wnt pathway, we found an as yet unidentified transcript of 24p3R. Our results indicate independent regulation of 24p3 expression by the Wnt pathway and by the intracellular iron level. Differential splicing of the 24p3R transcript, depending on the activation state of the Wnt pathway, may modify the function of 24p3.

Up to 10-20% of patients with Coronavirus Disease 2019 (COVID-19) develop a severe pulmonary disease due to immune dysfunction and cytokine dysregulation. However, the extracellular proteomic characteristics in respiratory tract of these critical COVID-19 still remains to be investigated. In the present study, we performed a quantitative proteomic analysis of the bronchoalveolar lavage fluid (BALF) from patients with critical COVID-19 and from non-COVID-19 controls. Our study identified 358 differentially expressed BALF proteins (p < 0.05), among which 41 were significantly changed after using the Benjamini-Hochberg correction (q < 0.05). The up-regulated signaling was found to be mainly involved in inflammatory signaling and response to oxidative stress. A series of increased extracellular factors including Tenascin-C (TNC), Mucin-1 (KL-6 or MUC1), Lipocalin-2 (LCN2), periostin (POSTN), Chitinase 3-like 1 (CHI3L1 or YKL40), S100A12, as well as the antigens including lymphocyte antigen 6D /E48 antigen (LY6D), CD9 antigen, CD177 antigen, prostate stem cell antigen (PSCA) were identified. Among which, the pro-inflammatory factor TNC and KL-6, that were further validated in serum of another thirty-nine COVID-19 patients and healthy controls, showing high potentials of being biomarkers or therapeutic candidates for COVID-19. This BALF proteome associated with COVID-19 would also be a valuable resource for researches on anti-inflammatory medication and understanding the molecular mechanisms of host response.

Keywords: Bronchoalveolar lavage fluid; COVID-19; KL-6; Quantitative proteomics; Tenascin-C.

OBJECTIVE

Lipocalin 2 (LCN2), an important mediator of a variety of cellular processes, is involved in regulating the inflammatory response, but its roles in different inflammatory diseases are controversial. Because the role of LCN2 in ocular inflammation has been unclear until now, we explored the function of LCN2 in lipopolysaccharide (LPS)-induced ocular inflammation in vivo and in vitro.

METHODS

Endotoxin-induced uveitis (EIU) was induced in male Sprague Dawley rats by the intravitreal injection of LPS. The expression and location of LCN2 in the retina were detected with western blotting and immunohistochemistry, respectively. We determined the clinical scores for anterior inflammation, quantified the infiltrated inflammatory cells, and measured the pro-inflammatory factors to determine the anti-inflammatory effects of LCN2 in EIU eyes. Cultured primary rat Müller cells were stimulated with LPS and the expression and secretion of LCN2 were measured with real-time PCR, western blotting, and an ELISA. After Müller cells were cotreated with LPS and LCN2 or PBS, the expression and secretion of TNF-α, IL-6, and MCP-1 were examined with realtime PCR, western blotting, and ELISAs. Western blotting and immunofluorescence were used to detect the phosphorylation and cellular distribution of nuclear factor kappaB (NF-κB) subunit p65.

RESULTS

In EIU, the expression of LCN2 was significantly upregulated in the retina, especially in the outer nuclear layer (mainly composed of Müller cells). LPS stimulation of cultured Müller cells also markedly elevated LCN2 expression. Intravitreal injection of LCN2 significantly reduced the clinical scores, inflammatory infiltration, and protein leakage in EIU, which correlated with the reduced levels of proinflammatory factors in the aqueous humor and retina. LCN2 treatment also reduced the expression and secretion of TNF-α, IL-6, and MCP-1 in LPS-stimulated Müller cells. LCN2 inhibited the inflammatory response by inhibiting the phosphorylation and translocation of NF-κB p65.

CONCLUSIONS

LCN2 protects against ocular inflammation, at least in part, by negatively regulating the activation of the NF-κB signaling pathway. LCN2 may be a promising anti-inflammatory therapy for ocular diseases, such as uveitis.

Mammalian sperm undergo multiple maturation steps after leaving the testis in order to become competent for fertilization, but the molecular mechanisms underlying this process remain unclear. In terms of identifying factors crucial for these processes in vivo, we found that lipocalin 2 (Lcn2), which is known as an innate immune factor inhibiting bacterial and malarial growth, can modulate sperm maturation. Most sperm that migrated to the oviduct of wild-type females underwent lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding, which are signatures of sperm maturation, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine to reinforce lipid raft reorganization via a PKA-dependent mechanism and promotes sperm to acquire fertility by facilitating cholesterol efflux. These observations imply that mammals possess a mode for sperm maturation in addition to the albumin-mediated pathway.

Acute kidney injury (AKI) can be fatal and is a well-defined risk factor for the development of chronic kidney disease. Group 2 innate lymphoid cells (ILC2s) are innate producers of type-2 cytokines and are critical regulators of homeostasis in peripheral organs. However, our knowledge of their function in the kidney is relatively limited. Recent evidence suggests that increasing ILC2 numbers by systemic administration of recombinant interleukin (IL)-25 or IL-33 protects against renal injury. Whilst ILC2s can be induced to protect against ischemic- or chemical-induced AKI, the impact of ILC2 deficiency or depletion on the severity of renal injury is unknown. Firstly, the phenotype and location of ILC2s in the kidney was assessed under homeostatic conditions. Kidney ILC2s constitutively expressed high levels of IL-5 and were located in close proximity to the renal vasculature. To test the functional role of ILC2s in the kidney, an experimental model of renal ischemia-reperfusion injury (IRI) was used and the severity of injury was assessed in wild-type, ILC2-reduced, ILC2-deficient, and ILC2-depleted mice. Surprisingly, there were no differences in histopathology, collagen deposition or mRNA expression of injury-associated (Lcn2), inflammatory (Cxcl1, Cxcl2, and Tnf) or extracellular matrix (Col1a1, Fn1) factors following IRI in the absence of ILC2s. These data suggest the absence of ILC2s does not alter the severity of renal injury, suggesting possible redundancy. Therefore, other mechanisms of type 2-mediated immune cell activation likely compensate in the absence of ILC2s. Hence, a loss of ILC2s is unlikely to increase susceptibility to, or severity of AKI.

Like many other organs, bone can act as an endocrine organ through the secretion of bone-specific hormones or "osteokines." At least two osteokines are implicated in the control of glucose and energy metabolism: osteocalcin (OCN) and lipocalin-2 (LCN2). OCN stimulates the production and secretion of insulin by the pancreatic β-cells, but also favors adaptation to exercise by stimulating glucose and fatty acid (FA) utilization by the muscle. Both of these OCN functions are mediated by the G-protein-coupled receptor GPRC6A. In contrast, LCN2 influences energy metabolism by activating appetite-suppressing signaling in the brain. This action of LCN2 occurs through its binding to the melanocortin 4 receptor (MC4R) in the paraventricular nucleus of the hypothalamus (PVN) and ventromedial neurons of the hypothalamus.

Lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL), a key antibacterial protein, is highly elevated in patients with end-stage liver disease that is often associated with bacterial infection. LCN2 is expressed at high levels in both hepatocytes and neutrophils; however, how hepatocyte-derived and neutrophil-derived LCN2 cooperate to combat bacterial infection remains unclear. Here, by studying hepatocyte-specific and myeloid-specific Lcn2 knockout mice in two models of systemic and local Klebsiella pneumoniae infections, we demonstrated that hepatocytes played a critical role in controlling systemic infection by secreting LCN2 protein into the circulation following intraperitoneal injection of bacteria, whereas neutrophils were more important in combating local lung infection by carrying LCN2 in their specific granules to the local infection site following intratracheal intubation of bacteria. Both hepatocyte-derived and myeloid cell-derived LCN2 were required against bacterial infection in the peritoneal cavity and liver necrotic areas following intraperitoneal injection of Klebsiella pneumoniae. LCN2/NGAL protein was detected in neutrophil extracellular traps (NETs) in activated neutrophils from mice and humans. Disruption of the Lcn2 gene in neutrophils abolished LCN2 on NETs, whereas deletion of this gene in hepatocytes did not affect LCN2 protein on NETs. Genetic deletion of the Lcn2 gene globally or specifically in neutrophils did not affect NET formation but reduced the bactericidal effect of NETs in vitro. Finally, NGAL-positive NETs were detected in the liver from patients with various types of liver diseases.

CONCLUSIONS

Both hepatocytes and neutrophils combat bacterial infection through the production of LCN2; extracellular LCN2 secreted by hepatocytes limits systemic bacterial infection, whereas neutrophils carry LCN2 protein to the local site and against local bacterial infection through NETs. (Hepatology 2018).

Lipocalin-2 (LCN2) is an antimicrobial protein and adipokine associated with insulin resistance, obesity and atherosclerotic disease. Psoriasis is a T-helper (Th)1/Th17-mediated, chronic inflammatory dermatosis related to metabolic syndromes and serum LCN2 levels are elevated in psoriatic patients. We examined the in vivo effects of LCN2 on topical imiquimod (IMQ)-induced psoriasiform skin in BALB/c mice and in vitro on human keratinocytes (KC). Clinically, i.p. injected LCN2 exacerbated erythema and scaling in IMQ-treated murine skin compared with phosphate-buffered saline injection alone, and it augmented interleukin (IL)-17A, IL-17F, IL-22, IL-23p19, IL-12p40, CCL20, tumor necrosis factor-α, chemokine (C-X-C motif) ligand (CXCL)1, CXCL2, DEFB4, DEFB14, LCN2 and S100A7 mRNA levels of IMQ-treated murine skin while it did not increase the mRNA levels of interferon-γ, IL-12p35 or CXCL10. LCN2 in synergy with IL-17 increased mRNA levels of CCL20, LCN2 and DEFB4A but not of CXCL10 in human KC in vitro. These results suggest that LCN2 enhances the expression of Th17 cytokines/chemokines and antimicrobial peptides in murine IMQ-treated psoriatic skin and KC. LCN2 may potentiate the development of psoriasis via the enhancement of Th17- and antimicrobial peptide-mediated inflammation.

Lipocalin-2 (LCN2) is a secreted glycoprotein linked to several physiological roles, including transporting hydrophobic ligands across cell membranes, modulating immune responses, maintaining iron homeostasis, and promoting epithelial cell differentiation. Although LNC2 is expressed at low levels in most human tissues, it is abundant in aggressive subtypes of cancer, including breast, pancreas, thyroid, ovarian, colon, and bile duct cancers. High levels of LCN2 have been associated with increased cell proliferation, angiogenesis, cell invasion, and metastasis. Moreover, LCN2 modulates the degradation, allosteric events, and enzymatic activity of matrix metalloprotease-9, a metalloprotease that promotes tumor cell invasion and metastasis. Hence, LCN2 has emerged as a potential therapeutic target against many cancer types. This review summarizes the most relevant findings regarding the expression, biological roles, and regulation of LCN2, as well as the proteins LCN2 interacts with in cancer. We also discuss the approaches to targeting LCN2 for cancer treatment that are currently under investigation, including the use of interference RNAs, antibodies, and gene editing.

Keywords: LCN2-MMP-9; NGAL; cancer; lipocalin 2; oncogene; siderophore.

Ferroptosis is a type of iron-dependent regulated cell death, representing an emerging disease-modulatory mechanism. Transcription factors play multiple roles in ferroptosis, although the key regulator for ferroptosis in iron metabolism remains elusive. Using NanoString technology, we identify NUPR1, a stress-inducible transcription factor, as a driver of ferroptosis resistance. Mechanistically, NUPR1-mediated LCN2 expression blocks ferroptotic cell death through diminishing iron accumulation and subsequent oxidative damage. Consequently, LCN2 depletion mimics NUPR1 deficiency with respect to ferroptosis induction, whereas transfection-enforced re-expression of LCN2 restores resistance to ferroptosis in NUPR1-deficient cells. Pharmacological or genetic blockade of the NUPR1-LCN2 pathway (using NUPR1 shRNA, LCN2 shRNA, pancreas-specific Lcn2 conditional knockout mice, or the small molecule ZZW-115) increases the activity of the ferroptosis inducer erastin and worsens pancreatitis, in suitable mouse models. These findings suggest a link between NUPR1-regulated iron metabolism and ferroptosis susceptibility.

T-helper-17 (Th17) cells are a subset of CD4+ T cells that can produce the cytokine interleukin (IL)-17 and play vital roles in protecting the host from bacterial and fungal infections, especially at the mucosal surface. These are abundant in the small intestinal lamina propria (SILP) and their differentiation are associated with the colonization of the intestinal flora. Segmented filamentous bacteria (SFB) drew the attention of researchers due to their unique ability to drive the accumulation of Th17 cells in the SI LP of mice. Recent work has highlighted that SFB used microbial adhesion-triggered endocytosis (MATE) to transfer SFB antigenic proteins into small intestinal epithelial cells (SI ECs) and modulate host immune homeostasis. However, which components of SFB are involved in this immune response process remains unclear. Here, we examined the roles of SFB flagellins in Th17 cells induction using various techniques, including ELISA, ELISPOT, and RNA-seq in vitro and in vivo. The results show that the immune function of SFB flagellins is similar to SFB, i.e., induces the appearance of CD4+ T helper cells that produce IL-17 and IL-22 (Th17 cells) in the SI LP. Furthermore, treatment of mice with SFB flagellins lead to a significant increase in the expression of genes associated with the IL-17 signaling pathway, such as IL-6, IL-1β, TNF-α, IL-17A, IL-17F, and IL-22. In addition, SFB flagellins have an intimate relationship with intestinal epithelial cells, influencing the expression of epithelial cell-specific genes such as Nos2, Duox2, Duoxa2, SAA3, Tat, and Lcn2. Thus, we propose that SFB flagellins play a significant role in the involvement of SFB in the induction of intestinal Th17 cells.