We have developed a highly sensitive immunoassay using graphene nano platelets (GNPs) for the rapid detection of human lipocalin-2 (LCN2) in plasma, serum, and whole blood. It has the dynamic range, linear range, limit of detection, and analytical sensitivity of 0.6 to 5120, 80 to 2560, 0.7, and 1pg/ml, respectively. It is the most sensitive assay for the detection of LCN2, which has 80-fold higher analytical sensitivity and 3-fold lesser immunoassay duration than the commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit. The functionalization of microtiter plate (MTP) with GNPs, dispersed in 3-aminopropyltriethoxysilane (APTES), provided the increased surface area that leads to higher immobilization density of capture antibodies. Moreover, the generation of free amino groups on MTP and GNPs by APTES enables the leach-proof covalent crosslinking of anti-human LCN2 capture antibody by its carboxyl groups using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as the heterobifunctional crosslinker. The anti-LCN2 antibody-bound MTPs were highly stable given that they did not show any significant decrease in their functional activity when stored at 4°C in 0.1M phosphate-buffered saline (PBS) for 8weeks. The developed immunoassay correlated well with the conventional ELISA, thereby demonstrating its high precision and potential utility for highly sensitive analyte detection in industrial and clinical settings.

OBJECTIVE

To compare lipocalin-2 (LCN2) levels in pre-eclamptic women with those in healthy pregnant women, and to determine whether there is a correlation between LCN2 levels and the severity of the disease.

METHODS

The study included 66 pregnant women: 22 healthy pregnant women (Group 1), 23 women with mild pre-eclampsia (Group 2), and 21 women with severe pre-eclampsia (Group 3). Pre-eclamptic women and normal controls were carefully matched for maternal age, gestational age, and body mass index (BMI). The maternal levels of plasma LCN2 were determined by enzyme-linked immunosorbent assay.

RESULTS

Plasma LCN2 levels in the pre-eclamptic group were significantly lower than those in the healthy control group (p < 0.05). Although plasma LCN2 level was lower in the severe compared to the mild pre-eclamptic group, the difference was not statistically significant (p>> 0.05). There was no significant correlation between LCN2 levels and the homeostasis model assessment of insulin resistance (HOMA-IR), BMI, triglyceride, gestational week at delivery, birth weight, and systolic and diastolic blood pressure in pre-eclamptic and healthy pregnant women (p>> 0.05).

CONCLUSIONS

Our results show that there are decreased concentrations of plasma LCN2 in pre-eclamptic subjects and this may indicate that LCN2 plays a role in the pathogenesis of pre-eclampsia. However, further experiments are needed to clarify this role.

Background: Informative serum biomarkers for monitoring inflammatory activity and treatment responses in axial spondyloarthritis (axSpA) are lacking. We assessed whether Lipocalin 2 (LCN2) and Oncostatin M (OSM), both having roles in inflammation and bone remodeling, may accurately reflect chronic joint inflammation and treatment response in axSpA. Previous reports in animal models showed involvement of LCN2 and OSM in joint/gut inflammation. We asked whether they also play a role in human axSpA.

Methods: We analyzed a longitudinal observational axSpA cohort (286 patients) with yearly clinical assessments and concurrent measurements of serum LCN2 and OSM (1204 serum samples) for a mean of 4 years. Biomarker levels were correlated with MRI scoring and treatment response.

Results: Persistent and transient elevation of LCN2 and OSM were observed in axSpA patients. Persistent elevation of LCN2 or OSM, but not CRP, correlated with sacroiliac joint (SIJ) MRI SPARCC scores (Pearson's correlation p = 0.0005 and 0.005 for LCN2 and OSM respectively), suggesting that LCN2/OSM outperforms CRP as reflective of SIJ inflammation. We observed both concordant and discordant patterns of LCN2 and OSM in relationship to back pain, the cardinal clinical symptom in axSpA. Twenty-six percent (73/286) of the patients remained both clinically and serologically active (CASA). Sixty percent (173/286) of the patients became clinically quiescent, with back pain resolved, but 53% (92/173) of them were serologically active (CQSA), indicating that pain control may not indicate control of joint inflammation, as reflected by positive MRI imaging of SIJ. With respect to treatment responses, transient elevation of LCN2 or OSM over time was predictive of better response to all treatments.

Conclusion: In axSpA, persistent LCN2 and/or OSM elevation reflects chronic SIJ inflammation and suboptimal treatment response. In our cohort, half of the currently deemed clinically quiescent patients with back pain resolved continued to demonstrate chronic joint inflammation. LCN2 and OSM profiling outperforms CRP as a predictive measure and provides an objective assessment of chronic local inflammation in axSpA patients.

Keywords: Axial spondyloarthritis; Joint inflammation; Lipocalin 2; MRI; Oncostatin M; Treatment response.

The commensal gut microbiota critically regulates immunomodulatory processes that influence normal skeletal growth and maturation. However, the influence of specific microbes on commensal gut microbiota osteoimmunoregulatory actions is unknown. We have shown previously that the commensal gut microbiota enhances T_HHHIl17a in ileum and IL17A in serum. SFB-colonized mice had an osteopenic trabecular bone phenotype, which was attributed to SFB actions suppressing osteoblastogenesis and enhancing osteoclastogenesis. Intriguingly, SFB-colonized mice had increased expression of proinflammatory chemokines and acute-phase reactants in the liver. Lipocalin-2 (LCN2), an acute-phase reactant and antimicrobial peptide, was substantially elevated in the liver and serum of SFB-colonized mice, which supports the notion that SFB regulation of commensal gut microbiota osteoimmunomodulatory actions are mediated in part through a gut-liver-bone axis. Proinflammatory T_HHIl17a in the gut and Lcn2 in the liver resulted in increased circulating levels of IL17A and LCN2. Recognizing that IL17A and LCN2 support osteoclastogenesis/suppress osteoblastogenesis, SFB actions impairing postpubertal skeletal development appear to be mediated through immunomodulatory effects in both the gut and liver. This research reveals that specific microbes critically impact commensal gut microbiota immunomodulatory actions regulating normal postpubertal skeletal growth and maturation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Rationale: Early pathogenesis of lung adenocarcinoma (LUAD) remains largely unknown. We found that, relative to wild-type littermates, the innate immunomodulator lipocalin-2 (Lcn2) was increased in normal airways from mice with knockout of the airway lineage gene Gprc5a (Gprc5a^-/-), and that are prone to developing inflammation and LUAD. Yet, the role of LCN2 in lung inflammation and LUAD is poorly understood. Objectives: Delineate the role of Lcn2 induction in LUAD pathogenesis. Methods: Normal airway brushings, uninvolved lung tissues and tumors from Gprc5a^-/- mice prior to and following tobacco carcinogen exposure were analyzed by RNA-sequencing. LCN2 mRNA was analyzed in public and in-house datasets of LUAD, lung squamous cancer (LUSC), chronic obstructive pulmonary disease (COPD) and LUAD/LUSC with COPD. LCN2 protein was immunohistochemically analyzed in a tissue microarray of 510 tumors. Temporal lung tumor development, gene expression programs and host immune responses were compared between Gprc5a^-/- and Gprc5a^-/-/Lcn2^-/- littermates. Measurements and Main Results: Lcn2 was progressively elevated during LUAD development and positively correlated with pro-inflammatory cytokines and inflammation gene sets. LCN2 was distinctively elevated in human LUADs, but not in LUSCs, relative to normal lungs and was associated with COPD among smokers and LUAD patients. Relative to Gprc5a^-/- mice, Gprc5a^-/-/Lcn2^-/- littermates exhibited significantly increased lung tumor development concomitant with reduced T cell abundance (CD4+) and richness, attenuated anti-tumor immune gene programs and increased immune cell expression of pro-tumor inflammatory cytokines. Conclusions: Augmented LCN2 expression is a molecular feature of COPD-associated LUAD and counteracts LUAD development in vivo by maintaining anti-tumor immunity.

Keywords: Chronic Obstructive; Immunity; Lung Neoplasms; Pulmonary Disease.

Dendritic polyglycerols (dPG), particularly dendritic polyglycerol sulfates (dPGS), have been intensively studied due to their intrinsic anti-inflammatory activity. As related to brain pathologies involving neuroinflammation, the current study examined if dPG and dPGS can (i) regulate neuroglial activation, and (ii) normalize the morphology and function of excitatory postsynaptic dendritic spines adversely affected by the neurotoxic 42 amino acid amyloid-β (Aβ42) peptide of Alzheimer disease (AD). The exact role of neuroglia, such as microglia and astrocytes, remains controversial especially their positive and negative impact on inflammatory processes in AD. To test dPGS effectiveness in AD models we used primary neuroglia and organotypic hippocampal slice cultures exposed to Aβ42 peptide. Overall, our data indicate that dPGS is taken up by both microglia and astrocytes in a concentration- and time-dependent manner. The mechanism of action of dPGS involves binding to Aβ42, i.e., a direct interaction between dPGS and Aβ42 species interfered with Aβ fibril formation and reduced the production of the neuroinflammagen lipocalin-2 (LCN2) mainly in astrocytes. Moreover, dPGS normalized the impairment of neuroglia and prevented the loss of dendritic spines at excitatory synapses in the hippocampus. In summary, dPGS has desirable therapeutic properties that may help reduce amyloid-induced neuroinflammation and neurotoxicity in AD.

Breast tumorigenesis is classically studied in mice by inoculating tumor cells in the fat pad, the adipose compartment of the mammary gland. Alternatively, the mammary ducts, which constitute the luminal mammary gland compartment, also provide a suitable inoculation site to induce breast cancer in murine models. The microenvironments in these compartments influence tumor cell progression, yet this effect has not been investigated in an immunocompetent context. Here, we compared both mammary gland compartments as distinct inoculation sites, taking into account the immunological aspect by inoculating 4T1 tumor cells in immunocompetent mice. Following tumor cell inoculation in the adipose compartment of non-pretreated/naive, hormonally pretreated/naive and non-pretreated/lactating mice, the primary tumors developed similarly. However, a slower onset of primary tumor growth was found after inoculations in the luminal compartment of non-pretreated/lactating mice. Despite this difference in tumor development rate, metastasis to the liver and lungs was equally observed and was accompanied by lymphatic spreading of tumor cells and progressive splenomegaly with both inoculation types. Chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) served as innovative biomarkers for disease progression showing increased levels in primary tumors and sera of the non-pretreated/lactating inoculation groups. A slower increase in circulating CHI3L1 but not LCN2 levels, was observed after inoculations in the luminal compartment which corroborated the slower tumor development at this inoculation site. Our results highlight the critical impact of different mammary gland compartments on tumor development in syngeneic murine models and support the use of novel tumor progression biomarkers in an immune-competent environment.

Lipocalin-2 (LCN2) is a secreted glycoprotein involved in several chronic inflammatory processes. Metabolic syndrome (MetS) and adult growth hormone deficiency (GHD) are known as chronic inflammatory conditions. The primary objective of this observational cross-sectional study was to compare LCN2 plasmatic levels in these clinical settings, whereas the secondary objective was to investigate any possible correlation between LCN2 and BMI and/or indexes of insulin sensitivity/resistance. Seventy-four patients were divided as follows: Group A, MetS (18 patients, 13 females and 5 males, mean ± SEM age 45.1 ± 4.11 years, BMI 31.22 ± 1.73 kg/m² ); Group B, total GHD (18 patients, 8 females and 10 males, age 52.44 ± 2.61 years, BMI 30.49 ± 1.87 kg/m² ); Group C, Partial GHD (pGHD; 19 patients, 13 females and 6 males, age 48.63 ± 2.19 years, BMI 29.11 ± 1.85 kg/m² ); Group D, Controls (19 patients, 13 females and 6males, age 40.26 ± 2.87 years, BMI 23.25 ± 0.95 kg/m² ). They were evaluated for glucose and insulin, HOMA-index, QUICKI-index, Total/low-density lipoprotein/high-density lipoprotein cholesterol, triglycerides, uric acid, IGF-1, and LCN2. LCN2 plasmatic levels were significantly increased in MetS, while no significant differences with controls were found in total and pGHD. LCN2 levels did not correlate with BMI. A significant positive correlation between LCN2 and HOMA-index was found in controls, while a trend-like, yet not significant, a positive correlation was observed in pGHD. Our data show an increase in LCN2 plasmatic levels in MetS. Different inflammatory patterns characterize MetS and GHD. The correlation between HOMA index and LCN2 in normal subjects and possibly in pGHD ones suggests a modulatory action of LCN2 on insulin resistance.

Dendritic spines, a special kind of structure in nerve cells, play a key role in performing cellular function. Structural abnormalities of the dendritic spine may contribute to synaptic dysfunction and have been implicated in memory formation. However, the molecular mechanisms that trigger dendritic spine loss remain unclear. Here, we show that the absence of dendritic cell factor 1 (Dcf1) appeared dendritic spines dysplasia, which in turn leads to the damage of learning and memory; in contrast, enhancing Dcf1 expression rescues dendritic spines morphology and function, indicating a pivotal role of Dcf1 in cellular function. Electrophysiological test indicates that there is a significant reduction in the frequency of miniature excitatory postsynaptic currents in Dcf1 -/- knockout (KO) mice. Subsequent to optogenetic ignition, we observed a weaker neuronal activation in Dcf1 KO mice, explaining the neural circuit cause. On molecular mechanism, we demonstrated an unprecedented discovery that Dcf1 triggers the dendritic spine and synaptic function through the recruitment of Lcn2 and activation of PSD95-NMDAR signaling. Removing this brake leads to memory damage. Our results highlight an unexpected regulatory mechanism of dendritic spine development and formation.

Mutations in the X-linked MECP2 gene are responsible for Rett syndrome (RTT), a severe neurological disorder. MECP2 is a transcriptional modulator that finely regulates the expression of many genes, specifically in the central nervous system. Several studies have functionally linked the loss of MECP2 in astrocytes to the appearance and progression of the RTT phenotype in a non-cell autonomous manner and mechanisms are still unknown. Here, we used primary astroglial cells from Mecp2-deficient (KO) pups to identify deregulated secreted proteins. Using a differential quantitative proteomic analysis, twenty-nine proteins have been identified and four were confirmed by Western blotting with new samples as significantly deregulated. To further verify the functional relevance of these proteins in RTT, we tested their effects on the dendritic morphology of primary cortical neurons from Mecp2 KO mice that are known to display shorter dendritic processes. Using Sholl analysis, we found that incubation with Lcn2 or Lgals3 for 48 h was able to significantly increase the dendritic arborization of Mecp2 KO neurons. To our knowledge, this study, through secretomic analysis, is the first to identify astroglial secreted proteins involved in the neuronal RTT phenotype in vitro, which could open new therapeutic avenues for the treatment of Rett syndrome.

Keywords: Mecp2; Rett syndrome; astrocytes; iTRAQ quantitative proteomic approach; neuronal arborization; secretome.

The central nervous system (CNS) responds to diverse neurologic injuries with a vigorous activation of astrocytes. In addition to their role in the maintenance of CNS homeostasis and neuronal function, astrocytes are thought to participate in the regulation of innate and adaptive immune responses in the CNS. Following antigen recognition, reactive astrocytes may participate in the initiation of innate immune responses, and modulate adaptive immune response leading to the recruitment of peripheral immune cells. Among activation, astrocytes undergo morphological changes and express several molecules, e.g., chemokines. Lipocalin 2 (LCN2) is involved in the control of innate immune responses, regulation of excess iron, and reactive oxygen production. Here, we investigated the influence of LCN2 on basic astrocytic functions linked to inflammatory responses. In vitro studies revealed a similar chemokine expression pattern in wild-type and Lcn2-deficient astrocyte cultures after treatment with lipopolysaccharides (LPS). Increased wound closure and morphological changes upon LPS treatment are independent of Lcn2 expression. We conclude that LCN2 is not necessary for basic astrocytic functions in the context of inflammation. However, CNS-derived LCN2 might have a regulatory effect on other cells, e.g., endothelial cells of the blood-brain barrier.

Keywords: Astrocyte; Infection; Lipocalin 2; Neuroinflammation.

Ischemic stroke causes rapid hypoxic damage to the core neural tissue which is followed by graded chronological tissue degeneration in the peri-infarct zone. The latter process is mainly triggered by neuroinflammation, activation of inflammasomes, proinflammatory cytokines, and pyroptosis. Besides microglia, astrocytes play an important role in the fine-tuning of the inflammatory network in the brain. Lipocalin-2 (LCN2) is involved in the control of innate immune responses, regulation of excess iron, and reactive oxygen production. In this study, we analyzed LCN2 expression in hypoxic rat brain tissue after ischemic stroke and in astrocyte cell cultures receiving standardized hypoxic treatment. Whereas no LCN2-positive cells were seen in sham animals, the number of LCN2-positive cells (mainly astrocytes) was significantly increased after stroke. In vitro studies with hypoxic cultured astroglia revealed that LCN2 expression is significantly increased after only 2 h, then further increased, followed by a stepwise decline. The expression pattern of several proinflammatory cytokines mainly followed that profile in wild type (WT) but not in cultured LCN2-deficient astrocytes. Our data revealed that astrocytes are an important source of LCN2 in the peri-infarct region under hypoxic conditions. However, we must also stress that brain-intrinsic LCN2 after the initial hypoxia period might come from other sources such as invaded immune cells and peripheral organs via blood circulation. In any case, secreted LCN2 might have an influence on peripheral organ functions and the innate immune system during brain hypoxia.

Chronic stress can impair the health of human brains. An important strategy that may prevent the accumulation of stress may be the consumption of functional foods. When senescence-accelerated mice prone 10 (SAMP10), a stress-sensitive strain, were loaded with stress using imposed male mouse territoriality, brain volume decreased. However, in mice that ingested theanine (6 mg/kg), the main amino acid in tea leaves, brain atrophy was suppressed, even under stress. On the other hand, brain atrophy was not clearly observed in a mouse strain that aged normally (Slc:ddY). The expression level of the transcription factor Npas4 (neuronal PAS domain protein 4), which regulates the formation and maintenance of inhibitory synapses in response to excitatory synaptic activity, decreased in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but increased in mice that ingested theanine. Lipocalin 2 (Lcn2), the expression of which increased in response to stress, was significantly high in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but not in mice that ingested theanine. These data suggest that Npas4 and Lcn2 are involved in the brain atrophy and stress vulnerability of SAMP10 mice, which are prevented by the consumption of theanine, causing changes in the expression of these genes.

Epidermal growth factor receptor (EGFR) activation and lipocalin-2 (Lcn2) expression are frequently observed in the same pathological contexts, such as cancers or chronic kidney disease (CKD). However, the significance of this association is unknown. Here, we describe the role of Lcn2 in regulating EGFR trafficking. We show that Lcn2 increases EGFR cell surface abundance and is required for transforming growth factor α (TGF-α)-induced EGFR recycling to the plasma membrane and sustained activation. Lcn2 binds to the intracellular domain of EGFR in late endosomal compartments and inhibits its lysosomal degradation. Consistently, Lcn2 enhances EGFR-induced cell migration after TGF-α stimulation. In vivo, Lcn2 gene inactivation prevents EGFR recycling to the plasma membrane in an experimental model of CKD. Remarkably, this is associated with a dramatic decrease of renal lesions. Together, our data identify Lcn2 as a key mediator of EGFR trafficking processes. Hence, therapeutic inhibition of Lcn2 may counteract the deleterious effect of EGFR activation.

In triple-negative breast cancer (TNBC), the lack of therapeutic markers and effective targeted therapies result in an incurable metastatic disease associated with a poor prognosis. Crosstalks within the tumor microenvironment (TME), including those between cancer and stromal cells, affect the tumor heterogeneity, growth, and metastasis. Previously, we have demonstrated that IL-6, IL-8, and CCL5 play a significant role in TNBC growth and metastasis. In this study, we performed a systematic analysis of cytokine factors secreted from four stromal components (fibroblasts, macrophages, lymphatic endothelial cells, and blood microvascular endothelial cells) induced by four TNBC cell types. Through bioinformatic analysis, we selected putative candidates of secreted factors from stromal cells, which are involved in EMT activity, cell proliferation, metabolism, and matrisome pathways. Among the candidates, LCN2, GM-CSF, CST3, IL-6, IL-8, and CHI3L1 are ranked highly. Significantly, Lipocalin-2 (LCN2) is upregulated in the crosstalk of stromal cells and four different TNBC cells. We validated the increase of LCN2 secreted from four stromal cells induced by TNBC cells. Using a specific LCN2 antibody, we observed the inhibition of TNBC cell growth and migration. Taken together, these results propose secreted factors as molecular targets to treat TNBC progression via crosstalk with stromal components.

Purpose: TP53 and the TGFβ pathway are major mediators of pancreatic cancer metastasis. The mechanisms by which they cause hematogenous metastasis have not been fully explored.Experimental Design:KPC (LSL-KRASG12D/+;LSL-Trp53R172H/+; Ptf1aCre/+) mice were generated, and the frequency and morphology of organ-specific hematogenous metastases compared with that seen in KPTC and KTC littermates (Tgfbr2+/-). Key findings were validated in primary cells from each genotype and samples of human pancreatic cancer liver metastases.Results: The frequency of hematogenous metastasis in KPTC mice was significantly lower than for KPC mice (41% vs. 68%, P < 0.05), largely due to a reduction in liver metastases. No differences were found between KPC and KPTC lung metastases, whereas liver metastases in KPTC mice showed a profound extravasation deficiency characterized by sinusoidal growth and lack of desmoplastic stroma. Analogous findings were confirmed in liver samples from patients indicating their clinical relevance. Portal vein colonization as a direct mode of access to the liver was observed in both mice and humans. Secretome analyses of KPC cells revealed an abundance of secreted prometastatic mediators including Col6A1 and Lcn2 that promoted early steps of metastatic colonization. These mediators were overexpressed in primary tumors but not metastases, suggesting that the ability to colonize is, in part, developed within the primary site, a phenomenon we refer to as the "Cinderella effect."Conclusions: These findings establish a novel paradigm for understanding pancreatic cancer metastasis and the observed clinical latencies of liver versus lung metastases specifically. Clin Cancer Res; 23(6); 1607-20. ©2016 AACR.

Chronic low-grade inflammation-induced insulin resistance is associated with neuroinflammation. Myeloid sirtuin1 (SIRT1) deficiency aggravates high-fat diet (HFD)-induced insulin resistance. However, the function of myeloid-specific SIRT1 in the hippocampus of obese mice is largely unknown. To address this question, we fed myeloid SIRT1 knockout (KO) mice a HFD for 40 weeks. We found that HFD-fed SIRT1 KO mice had increased insulin resistance and macrophage infiltration in adipose tissue than wild type (WT) mice. Levels of HFD-induced lipocalin-2 (LCN2) were lower in SIRT1 KO mice than in WT. HFD-induced hippocampal LCN2 expression was lower in HFD-fed SIRT1 KO mice than in WT. Hippocampal acetylation of nuclear factor-κB (NF-κB) and amyloid precursor protein levels were higher in HFD-fed SIRT1 KO mice than in HFD-fed WT mice. Taken together, our results suggest that targeted induction of the anti-inflammatory effects of SIRT1 and LCN2 may help prevent obesity-associated insulin resistance and neuroinflammation.

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella type IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a Coxiella T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild-type (WT) bacteria, suggesting that Coxiella T4BSS effector proteins downregulate the expression of these genes. In addition, the interleukin-17 (IL-17) signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during Coxiella infection is unknown. We found that IL-17 kills intracellular Coxiella in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed the increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT- or mock-infected cells, including the proinflammatory cytokine genes Il1a, Il1b, and Tnfa, the chemokine genes Cxcl2 and Ccl5, and the antimicrobial protein gene Lcn2 We further confirmed that the Coxiella T4BSS downregulates macrophage CXCL2/macrophage inflammatory protein 2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that Coxiella downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response.

A comparative analysis of inflammation between solid organs following donor brain death (BD) is still lacking and the detailed influence of BD accelerating ischaemia-reperfusion injury (IRI) post-transplantation remains to be addressed. Applying a murine model of BD, we demonstrated that 4 h after BD organs were characterized by distinct inflammatory expression patterns. For instance, lipocalin 2 (LCN2), a marker of acute kidney injury, was selectively induced in BD livers but not in kidneys. BD further resulted in significantly reduced frequencies of CD3(+) CD4(+) , CD3(+) CD8(+) T cells and NKp46(+) NK cells in the liver, whereas BD kidneys and hearts were characterized by significantly lower frequencies of conventional dendritic cells (cDCs). Syngeneic models of kidney (KTx) and heart transplantation (HTx) illustrated stronger gene expression in engrafted BD hearts only, but 20 h post-transplantation both organs displayed comparable intragraft lymphocyte frequencies, except for NK cells and graft function. Moreover, the complement factor C3d deposit detected in small vessels and capillaries in cardiac syngrafts did not significantly differ between BD and sham-transplanted groups. Finally, no further influence of donor BD on graft survival was detected in an allogeneic heart transplantation setting (C57BL/6 grafts into BALB/c recipients). We show for the first time that BD organs are characterized by a varying inflammatory profile; however, BD does not accelerate IRI in syngeneic KTx and HTx.

Klebsiella pneumoniae (Kp), one of the most common causes of healthcare-associated infections, increases patient morbidity, mortality, and hospitalization costs. Kp must acquire nutrients from the host for successful infection; however, the host is able to prevent bacterial nutrient acquisition through multiple systems. This includes the innate immune protein lipocalin 2 (Lcn2), which prevents Kp iron acquisition. To identify novel Lcn2-dependent Kp factors that mediate evasion of nutritional immunity during lung infection, we undertook an InSeq study using a pool of >20,000 transposon mutants administered to Lcn2+/+ and Lcn2-/- mice. Comparing transposon mutant frequencies between mouse genotypes, we identified the Kp citrate synthase, GltA, as potentially interacting with Lcn2, and this novel finding was independently validated. Interestingly, in vitro studies suggest that this interaction is not direct. Given that GltA is involved in oxidative metabolism, we screened the ability of this mutant to use a variety of carbon and nitrogen sources. The results indicated that the gltA mutant has a distinct amino acid auxotrophy rendering it reliant upon glutamate family amino acids for growth. Deletion of Lcn2 from the host leads to increased amino acid levels in bronchioloalveolar lavage fluid, corresponding to increased fitness of the gltA mutant in vivo and ex vivo. Accordingly, addition of glutamate family amino acids to Lcn2+/+ bronchioloalveolar lavage fluid rescued growth of the gltA mutant. Using a variety of mouse models of infection, we show that GltA is an organ-specific fitness factor required for complete fitness in the spleen, liver, and gut, but dispensable in the bloodstream. Similar to bronchioloalveolar lavage fluid, addition of glutamate family amino acids to Lcn2+/+ organ lysates was sufficient to rescue the loss of gltA. Together, this study describes a critical role for GltA in Kp infection and provides unique insight into how metabolic flexibility impacts bacterial fitness during infection.

Lipocalin-2 (LCN2), a protein secreted mainly by activated neutrophils, has been associated with neurodegeneration, obesity, and inflammatory responses. Serum LCN2 concentration has been reported elevated in patients with psoriasis, but lower in patients with atopic dermatitis (AD). Spinal astrocyte-derived LCN2 was found to be involved in enhancement of itch in a mouse model of AD. However, the relationship between LCN2 and itch in patients with psoriasis has not been determined. Objective. This study examined the correlation between serum LCN2 levels and the degrees of itch in patients with psoriasis.

Serum LCN2 concentrations were measured by enzyme-linked immunosorbent assays (ELISA) in patients with psoriasis and AD and in healthy controls. The degree of itch was assessed using a visual analog scale (VAS), and disease severity was determined by measuring psoriasis area and severity index (PASI) and scoring atopic dermatitis (SCORAD). Correlations among serum LCN2 level, VAS, PASI, and SCORAD were analyzed statistically. We further examined the serum LCN levels in psoriasis patients before and after biological treatment.Serum LCN2 concentrations were significantly higher in patients with psoriasis and AD than those in healthy controls. In patients with psoriasis, serum LCN2 concentrations were significantly correlated with VAS, but not with PASI. In contrast, serum LCN2 concentrations did not correlate with VAS or SCORAD in patients with AD. Serum LCN2 levels in psoriasis patients significantly decreased after the biological treatment along with improvement of VAS.Serum LCN2 concentration is associated with the degree of itch in patients with psoriasis, suggesting that serum LCN2 may be a useful clinical marker for itch in psoriasis.