Sphingosine kinases (SPHK) generate the sphingolipid sphingosine-1-phosphate, which, among other functions, is a potent regulator of inflammation. While SPHK1 produces S1P to promote inflammatory signaling, the role of SPHK2 is unclear due to divergent findings in studies utilizing gene depletion versus inhibition of catalytic activity. We sought to clarify how SPHK2 affects inflammatory signaling in human macrophages, which are main regulators of inflammation. SPHK2 expression and activity were rapidly decreased within 6 h upon stimulating primary human macrophages with lipopolysaccharide (LPS), but was upregulated after 24 h. At 24 h following LPS stimulation, targeting SPHK2 with the inhibitor ABC294640, a specific siRNA or by using Sphk2-/- mouse peritoneal macrophages increased inflammatory cytokine production. Downregulation of SPHK2 in primary human macrophages within 6 h of LPS treatment was blocked by inhibiting autophagy. SPHK2 overexpression or inhibiting autophagy 6 h after human macrophage activation with LPS suppressed inflammatory cytokine release. Mechanistically, SPHK2 suppressed LPS-triggered NF-κB activation independent of its catalytic activity and prevented increased mitochondrial ROS formation downstream of LPS. In conclusion, SPHK2 is an anti-inflammatory protein in human macrophages that is inversely coupled to inflammatory cytokine production. This needs consideration when targeting SPHK2 with specific inhibitors.

NF-E2-related factor 2 (Nrf2), known to protect against reactive oxygen species, has recently been reported to resolve acute inflammatory responses in activated macrophages. Consequently, disruption of Nrf2 promotes a proinflammatory macrophage phenotype. In the current study, we addressed the impact of this macrophage phenotype on CD8(+) T cell activation by using an antigen-driven coculture model consisting of Nrf2(-/-) and Nrf2(+/+) bone marrow-derived macrophages (BMDMΦ) and transgenic OT-1 CD8(+) T cells. OT-1 CD8(+) T cells encode a T cell receptor that specifically recognizes MHC class I-presented ovalbumin OVA(257-264) peptide, thereby causing a downstream T cell activation. Interestingly, coculture of OVA(257-264)-pulsed Nrf2(-/-) BMDMΦ with transgenic OT-1 CD8(+) T cells attenuated CD8(+) T cell activation, proliferation, and cytotoxic function. Since the provision of low-molecular-weight thiols such as glutathione (GSH) or cysteine (Cys) by macrophages limits antigen-driven CD8(+) T cell activation, we quantified the amounts of intracellular and extracellular GSH and Cys in both cocultures. Indeed, GSH levels were strongly decreased in Nrf2(-/-) cocultures compared to wild-type counterparts. Supplementation of thiols in Nrf2(-/-) cocultures via addition of glutathione ester, N-acetylcysteine, β-mercaptoethanol, or cysteine itself restored T cell proliferation as well as cytotoxicity by increasing intracellular GSH. Mechanistically, we identified two potential Nrf2-regulated genes involved in thiol synthesis in BMDMΦ: the cystine transporter subunit xCT and the modulatory subunit of the GSH-synthesizing enzyme γ-GCS (GCLM). Pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of the cystine antiporter xCT in Nrf2(+/+) BMDMΦ mimicked the effect of Nrf2(-/-) BMDMΦ on CD8(+) T cell function. Our findings demonstrate that reduced levels of GCLM as well as xCT in Nrf2(-/-) BMDMΦ limit GSH availability, thereby inhibiting antigen-induced CD8(+) T cell function.

Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of inflammatory leukotrienes. 5-LO gene expression is mainly restricted to B cells and cells of myeloid origin. It is known that basal 5-lipoxygenase promoter activity is regulated by DNA methylation. In this study we investigated the impact of the DNA methylation status of the 5-LO promoter on its activity and the role of methyl DNA binding proteins (MBDs) in transcriptional silencing of the 5-LO promoter. Using ChIP assays, we found that the methyl-DNA binding proteins MBD1, MBD2 and MeCP2 bind to the methylated 5-LO core promoter in U937 cells. Knock down of each of the MBDs upregulates 5-LO mRNA expression in U937 cells indicating that these proteins are involved in silencing of the 5-LO gene. In reporter gene assays with in vitro methylated 5-LO promoter constructs, the extent of 5-LO promoter methylation inversely correlated with its activity. Furthermore, we found that MBD1 overexpression repressed 5-LO promoter activity when the CpG sites at the Sp1 binding site close to the transcriptional start site (GC4) were methylated. Gel shift data indicate that recruitment of Sp1 to this binding site is prevented by methylation.

Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases.

PPARγ is a pharmacological target in inflammatory and metabolic diseases. Upon agonistic treatment or following antagonism, binding of co-factors is altered, which consequently affects PPARγ-dependent transactivation as well as its DNA-independent properties. Therefore, establishing techniques to characterize these interactions is an important issue in living cells. Methods: Using the FRET pair Clover/mRuby2, we set up a flow cytometry-based FRET assay by analyzing PPARγ1 binding to its heterodimerization partner RXRα. Analyses of PPARγ-reporter and co-localization studies by laser-scanning microscopy validated this system. Refining the system, we created a new readout to distinguish strong from weak interactions, focusing on PPARγ-binding to the co-repressor N-CoR2. Results: We observed high FRET in cells expressing Clover-PPARγ1 and mRuby2-RXRα, but no FRET when cells express a mRuby2-RXRα deletion mutant, lacking the PPARγ interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-ΔID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPARγ1, nor in murine J774A.1 macrophages. In HEK293T cells, binding was even lower in comparison to N-CoR2 constructs in which domains established to mediate interaction with PPARγ binding are deleted. These data suggest a possible role of N-CoR2-ΔID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPARγ1, we determined the effect of pharmacological treatment on FRET intensity. Therefore, we calculated flow cytometry-based FRET efficiencies based on our flow cytometry data. As with PPARγ antagonism, PPARγ agonist treatment did not prevent binding of N-CoR2. Conclusion: Our system allows the close determination of protein-protein interactions with a special focus on binding intensity, allowing this system to characterize the role of protein domains as well as the effect of pharmacological agents on protein-protein interactions.

Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option. Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection. Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers. Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.

The bioactive lipid sphingosine-1-phosphate (S1P), along with its receptors, modulates lymphocyte trafficking and immune responses to regulate skin inflammation. Macrophages are important in the pathogenesis of psoriasiform skin inflammation and express various S1P receptors. How they respond to S1P in skin inflammation remains unknown. We show that myeloid specific S1P receptor 1 (S1PR1) deletion enhances early inflammation in a mouse model of imiquimod-induced psoriasis, without altering the immune cell infiltrate. Mechanistically, myeloid S1PR1 deletion altered the formation of IL-1β, VEGF-A, and VEGF-C, and their receptors' expression in psoriatic skin, which subsequently lead to reciprocal regulation of neoangiogenesis and neolymphangiogenesis. Experimental findings were corroborated in human clinical datasets and in knockout macrophages in vitro. Increased blood vessel but reduced lymph vessel density may explain the exacerbated inflammatory phenotype in conditional knockout mice. These findings assign a novel role to macrophage S1PR1 and provide a rationale for therapeutically targeting local S1P during skin inflammation.

The peroxisome proliferator-activated receptor (PPARγ) is a central mediator of cellular lipid metabolism and immune cell responses during inflammation. This is facilitated by its role as a transcription factor as well as a DNA-independent protein interaction partner. We addressed how the cellular redox milieu in the cytosol and the nucleus of lipopolysaccharide (LPS)/interferon-γ- (IFNγ-) and interleukin-4- (IL4-) polarized macrophages (MΦ) initiates posttranslational modifications of PPARγ, that in turn alter its protein function. Using the redox-sensitive GFP2 (roGFP2), we validated oxidizing and reducing conditions following classical and alternative activation of MΦ, while the redox status of PPARγ was determined via mass spectrometry. Cysteine residues located in the zinc finger regions (amino acid fragments AA 90-115, AA 116-130, and AA 160-167) of PPARγ were highly oxidized, accompanied by phosphorylation of serine 82 in response to LPS/IFNγ, whereas IL4-stimulation provoked minor serine 82 phosphorylation and less cysteine oxidation, favoring a reductive milieu. Mutating these cysteines to alanine to mimic a redox modification decreased PPARγ-dependent reporter gene transactivation supporting a functional shift of PPARγ associated with the MΦ phenotype. These data suggest distinct mechanisms for regulating PPARγ function based on the redox state of MΦ.

Prostaglandin E2 (PGE2) favors multiple aspects of tumor development and immune evasion. Therefore, microsomal prostaglandin E synthase (mPGES-1/-2), is a potential target for cancer therapy. We explored whether inhibiting mPGES-1 in human and mouse models of breast cancer affects tumor-associated immunity. A new model of breast tumor spheroid killing by human PBMCs was developed. In this model, tumor killing required CD80 expression by tumor-associated phagocytes to trigger cytotoxic T cell activation. Pharmacological mPGES-1 inhibition increased CD80 expression, whereas addition of PGE2, a prostaglandin E2 receptor 2 (EP2) agonist, or activation of signaling downstream of EP2 reduced CD80 expression. Genetic ablation of mPGES-1 resulted in markedly reduced tumor growth in PyMT mice. Macrophages of mPGES-1(-/-) PyMT mice indeed expressed elevated levels of CD80 compared to their wildtype counterparts. CD80 expression in tumor-spheroid infiltrating mPGES-1(-/-) macrophages translated into antigen-specific cytotoxic T cell activation. In conclusion, mPGES-1 inhibition elevates CD80 expression by tumor-associated phagocytes to restrict tumor growth. We propose that mPGES-1 inhibition in combination with immune cell activation might be part of a therapeutic strategy to overcome the immunosuppressive tumor microenvironment.

NADPH oxidase activation in either RAW264.7 cells or peritoneal macrophages (PM) derived from PPARγ wild-type mice increased reactive oxygen species (ROS) formation, caused PPARγ activation, heme oxygenase-1 (HO-1) induction, and concomitant IFN-β expression. In macrophages transduced with a dominant negative (d/n) mutant of PPARγ (RAW264.7 AF2) as well as PPARγ negative PM derived from Mac-PPARγ-KO mice, NADPH oxidase-dependent IFN-β expression was attenuated. As the underlying mechanism, we noted decreased HO-1 mRNA stability in RAW264.7 AF2 cells as well as PPARγ negative PM, compared to either parent RAW264.7 cells or wild-type PM. Assuming mRNA stabilization of HO-1 by PPARγ we transfected macrophages with a HO-1 3'-UTR reporter construct. The PPARγ agonist rosiglitazone significantly up-regulated luciferase expression in RAW264.7 cells, while it remained unaltered in RAW264.7 AF2 macrophages. Deletion of each of two AU-rich elements in the 3'-UTR HO-1 decreased luciferase activity in RAW264.7 cells. Using LPS as a NADPH oxidase activator, PM from Mac-PPARγ-KO mice showed a decreased HO-1 mRNA half-life in vitro and in vivo compared to PPARγ wild-type mice. These data identified a so far unappreciated role of PPARγ in stabilizing HO-1 mRNA, thus, contributing to the expression of the HO-1 target gene IFN-β.

Interactions between tumor cells and cells in the microenvironment contribute to tumor development and metastasis. The spatial arrangement of individual cells in relation to each other influences the likelihood of whether and how these cells interact with each other.

An altered lipidome in tumors may affect not only tumor cells themselves but also their microenvironment. In this study, a lipidomics screen reveals increased amounts of phosphatidylserine (PS), particularly ether-PS (ePS), in murine mammary tumors compared with normal tissue. PS was produced by phosphatidylserine synthase 1 (PTDSS1), and depletion of Ptdss1 from tumor cells in mice reduced ePS levels accompanied by stunted tumor growth and decreased tumor-associated macrophage (TAM) abundance. Ptdss1-deficient tumor cells exposed less PS during apoptosis, which was recognized by the PS receptor MERTK. Mammary tumors in macrophage-specific Mertk-/- mice showed similarly suppressed growth and reduced TAM infiltration. Transcriptomic profiles of TAMs from Ptdss1-knockdown tumors and Mertk-/- TAMs revealed that macrophage proliferation was reduced when the Ptdss1/Mertk pathway was targeted. Moreover, PTDSS1 expression correlated positively with TAM abundance but negatively with breast carcinoma patient survival. PTDSS1 thus may be a target to modify tumor-promoting inflammation.

Good science in translational research requires good animal welfare according to the principles of 3Rs. In many countries, determining animal welfare is a mandatory legal requirement, implying a categorization of animal suffering, traditionally dominated by subjective scorings. However, how such methods can be objectified and refined to compare impairments between animals, subgroups, and animal models remained unclear. Therefore, we developed the RELative Severity Assessment (RELSA) procedure to establish an evidence-based method based on quantitative outcome measures such as body weight, burrowing behavior, heart rate, heart rate variability, temperature, and activity to obtain a relative metric for severity comparisons. The RELSA procedure provided the necessary framework to get severity gradings in TM-implanted mice, yielding four distinct RELSA thresholds L1<0.27, L2<0.59, L3<0.79, and L4<3.45. We show further that severity patterns in the contributing variables are time and model-specific and use this information to obtain contextualized between animal-model and subgroup comparisons with the severity of sepsis > surgery > restraint stress > colitis. The bootstrapped 95% confidence intervals reliably show that RELSA estimates are conditionally invariant against missing information but precise in ranking the quantitative severity information to the moderate context of the transmitter-implantation model. In conclusion, we propose the RELSA as a validated tool for an objective, computational approach to comparative and quantitative severity assessment and grading. The RELSA procedure will fundamentally improve animal welfare, data quality, and reproducibility. It is also the first step toward translational risk assessment in biomedical research.

Inflammatory conditions and oxidative stress contribute to the development of atherosclerosis. Nuclear factor E2-related factor 2 (Nrf2) is a redox-sensitive transcription factor known for its antioxidant, anti-inflammatory, and, thus, cell-protective properties. Its role in effecting a deactivated state of oxidized low-density lipoprotein (oxLDL)-generated foam cell macrophages (FCMs), a prevailing cellular phenotype of atherosclerotic lesions, has not been investigated yet. In this study RAW264.7- or mouse peritoneal macrophage-derived FCMs showed reduced mRNA expression of proinflammatory cytokines such as IL-1β and IL-6 and an attenuated production of reactive oxygen species (ROS), as analyzed by hydroethidine in response to lipopolysaccharide (LPS) and compared to LPS-treated control macrophages. In peritoneal FCMs from Nrf2-/- mice (C57BL/6J), the LPS-induced proinflammatory response was restored. OxLDL induced heme oxygenase (HO)-1, which was Nrf2-dependent, and inhibition of HO-1 activity in FCMs using zinc protoporphyrin-IX allowed the cells to regain a proinflammatory phenotype. Mechanistically, oxLDL attenuated ROS-dependent activation of CCAAT/enhancer binding protein (C/EBP) family members in FCMs, thereby reducing cytokine expression. Thus, in FCMs the Nrf2/HO-1 axis intervenes in LPS signaling. ROS production is impaired, C/EBP transactivation is reduced, and consequently the expression of proinflammatory mediators is attenuated, thereby shaping a desensitized FCM phenotype. This macrophage phenotype may be important for the progression of atherosclerosis.

Metastasis is the primary cause of cancer death. The inflammatory tumor microenvironment contributes to metastasis, for instance, by recruiting blood and lymph vessels. Among tumor-infiltrating immune cells, tumor-associated macrophages (TAMs) take a center stage in promoting both tumor angiogenesis and metastatic spread. We found that genetic deletion of the S1P receptor 1 (S1pr1) alone in CD11bhi CD206+ TAMs infiltrating mouse breast tumors prevents pulmonary metastasis and tumor lymphangiogenesis. Reduced lymphangiogenesis was also observed in the nonrelated methylcholanthrene-induced fibrosarcoma model. Transcriptome analysis of isolated TAMs from both entities revealed reduced expression of the inflammasome component Nlrp3 in S1PR1-deficient TAMs. Macrophage-dependent lymphangiogenesis in vitro was triggered upon inflammasome activation and required both S1PR1 signaling and IL-1β production. Finally, NLRP3 expression in tumor-infiltrating macrophages correlated with survival, lymph node invasion, and metastasis of mammary carcinoma patients. Conceptually, our study indicates an unappreciated role of the NLRP3 inflammasome in promoting metastasis via the lymphatics downstream of S1PR1 signaling in macrophages.

CD69 is a transmembrane lectin that can be expressed on most hematopoietic cells. In monocytes, it has been functionally linked to the 5-lipoxygenase pathway in which the leukotrienes, a class of highly potent inflammatory mediators, are produced. However, regarding CD69 gene expression and its regulatory mechanisms in monocytes, only scarce data are available. Here, we report that CD69 mRNA expression, analogous to that of 5-lipoxygenase, is induced by the physiologic stimuli transforming growth factor-β (TGF-β) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in monocytic cells. Comparison with T- and B-cell lines showed that the effect was specific for monocytes. CD69 expression levels were increased in a concentration-dependent manner, and kinetic analysis revealed a rapid onset of mRNA expression, indicating that CD69 is a primary TGF-β/1α,25(OH)2D3 target gene. PCR analysis of different regions of the CD69 mRNA revealed that de novo transcription was initiated and proximal and distal parts were induced concomitantly. In common with 5-lipoxygenase, no activation of 0.7 kb or ∼2.3 kb promoter fragments by TGF-β and 1α,25(OH)2D3 could be observed in transient reporter assays for CD69. Analysis of mRNA stability using a transcription inhibitor and a 3'UTR reporter construct showed that TGF-β and 1α,25(OH)2D3 do not influence CD69 mRNA stability. Functional knockdown of Smad3 clearly demonstrated that upregulation of CD69 mRNA, in contrast to 5-LO, depends on Smad3. Comparative studies with different inhibitors for mitogen activated protein kinases (MAPKs) revealed that MAPK signalling is involved in CD69 gene regulation, whereas 5-lipoxygenase gene expression was only partly affected. Mechanistically, we found evidence that CD69 gene upregulation depends on TAK1-mediated p38 activation. In summary, our data indicate that CD69 gene expression, conforming with 5-lipoxygenase, is regulated monocyte-specifically by the physiologic stimuli TGF-β and 1α,25(OH)2D3 on mRNA level, although different mechanisms account for the upregulation of each gene.

So far, there is only limited information about the regulation of the endogenous synthesis of hydrogen sulfide (H(2) S), an important gaseous signalling molecule. This study was done to evaluate the redox-dependent signalling events that regulate the expression of the H(2) S synthesising enzyme cystathionine-γ-lyase (CSE) in rat mesangial cells.

Recently, we provided evidence that PKCalpha depletion in monocytes/macrophages contributes to cellular desensitization during sepsis. We demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma) agonists dose dependently block PKCalpha depletion in response to the diacylglycerol homologue PMA in RAW 264.7 and human monocyte-derived macrophages. In these cells, we observed PPARgamma-dependent inhibition of nuclear factor-kappaB (NF-kappaB) activation and TNF-alpha expression in response to PMA. Elucidating the underlying mechanism, we found PPARgamma1 expression not only in the nucleus but also in the cytoplasm. Activation of PPARgamma1 wild type, but not an agonist-binding mutant of PPARgamma1, attenuated PMA-mediated PKCalpha cytosol to membrane translocation. Coimmunoprecipitation assays pointed to a protein-protein interaction of PKCalpha and PPARgamma1, which was further substantiated using a mammalian two-hybrid system. Applying PPARgamma1 mutation and deletion constructs, we identified the hinge helix 1 domain of PPARgamma1 that is responsible for PKCalpha binding. Therefore, we conclude that PPARgamma1-dependent inhibition of PKCalpha translocation implies a new model of macrophage desensitization.

Peroxisome proliferator-activated receptor gamma (PPARγ) modulators have found wide application for the treatment of cancers, metabolic disorders and inflammatory diseases. Contrary to PPARγ agonists, PPARγ antagonists have been much less studied and although they have shown immunomodulatory effects, there is still no therapeutically useful PPARγ antagonist on the market. In contrast to non-competitive, irreversible inhibition caused by 2-chloro-5-nitrobenzanilide (GW9662), the recently described (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl)benzyl)oxy)-benzylidene)-hexanoic acid (MTTB, T-10017) is a promising prototype for a new class of PPARγ antagonists. It exhibits competitive antagonism against rosiglitazone mediated activation of PPARγ ligand binding domain (PPARγLBD) in a transactivation assay in HEK293T cells with an IC50 of 4.3 µM against 1 µM rosiglitazone. The aim of this study was to investigate the structure-activity relationships (SAR) of the MTTB scaffold focusing on improving its physicochemical properties. Through this optimization, 34 new derivatives were prepared and characterized. Two new potent compounds (T-10075 and T-10106) with much improved drug-like properties and promising pharmacokinetic profile were identified.

During the course of sepsis in critically ill patients, kidney dysfunction and damage are among the first events of a complex scenario toward multi-organ failure and patient death. Acute kidney injury triggers the release of lipocalin-2 (Lcn-2), which is involved in both renal injury and recovery. Taking into account that Lcn-2 binds and transports iron with high affinity, we aimed at clarifying if Lcn-2 fulfills different biological functions according to its iron-loading status and its cellular source during sepsis-induced kidney failure. We assessed Lcn-2 levels both in serum and in the supernatant of short-term cultured renal macrophages (MΦ) as well as renal tubular epithelial cells (TEC) isolated from either Sham-operated or cecal ligation and puncture (CLP)-treated septic mice. Total kidney iron content was analyzed by Perls' staining, while Lcn-2-bound iron in the supernatants of short-term cultured cells was determined by atomic absorption spectroscopy. Lcn-2 protein in serum was rapidly up-regulated at 6 h after sepsis induction and subsequently increased up to 48 h. Lcn-2-levels in the supernatant of TEC peaked at 24 h and were low at 48 h with no change in its iron-loading. In contrast, in renal MΦ Lcn-2 was low at 24 h, but increased at 48 h, where it mainly appeared in its iron-bound form. Whereas TEC-secreted, iron-free Lcn-2 was associated with renal injury, increased MΦ-released iron-bound Lcn-2 was linked to renal recovery. Therefore, we hypothesized that both the cellular source of Lcn-2 as well as its iron-load crucially adds to its biological function during sepsis-induced renal injury.