Macrophage-Derived Iron-Bound Lipocalin-2 Correlates with Renal Recovery Markers Following Sepsis-Induced Kidney Damage.

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.

Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells.

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.

Tolerizing CTL by Sustained Hepatic PD-L1 Expression Provides a New Therapy Approach in Mouse Sepsis.

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.

Elevated intrathymic sphingosine-1-phosphate promotes thymus involution during sepsis.

Sepsis mouse models revealed thymus atrophy, characterised by decreased thymus weight and loss of thymocytes due to apoptosis. Mice suffered from lymphopenia, a lack of T cells in the periphery, which attenuates their ability to fight against recurring and secondary infections during sepsis progression. Key players in thymus atrophy are IL-6, which is directly involved in thymus involution, and the sphingosine-1-phosphate - sphingosine-1-phosphate receptor 1 signaling, influencing thymocytes emigration. In healthy individuals a sphingosine-1-phosphate (S1P) gradient from lymphoid organs to the circulatory system serves as signal for mature T cell egress. In the present study we investigated, whether inhibition of S1P generation improves thymus involution. In sepsis, induced by cecal ligation and puncture (CLP), S1P in the thymus increased, while it decreased in serum, thus disrupting the naturally occurring S1P gradient. As a potential source of S1P we identified increased numbers of apoptotic cells in the thymic cortex of septic mice. Pharmacological inhibition of the S1P generating sphingosine kinases, by 4- [[4-(4-Chlorophenyl)-2-thiazolyl]amino]phenol (SK I-II), administered directly following CLP, prevented thymus atrophy. This was reflected by lymphocytosis, diminished apoptosis, decreased IL-6 expression, and an unaltered thymus weight. In addition SK I-II-treatment preserved the S1P balance and prevented S1P-dependent internalization of the sphingosine-1-phosphate receptor 1. Our data suggest that inhibition of sphingosine kinase and thus, S1P generation during sepsis restores thymic T cell egress, which might improve septic outcome.

Apoptotic Diminution of Immature Single and Double Positive Thymocyte Subpopulations Contributes to Thymus Involution During Murine Polymicrobial Sepsis.

To generate and maintain functional T-cell receptor diversity, thymocyte development is tightly organized. Errors in this process may have dramatic consequences, provoking, for example, autoimmune diseases. Probably for this reason, the thymus reacts to septic stress with involution, decreasing the numbers of thymocytes. Because it is still unclear which thymocyte subpopulation contributes to thymus involution and whether thymocyte emigration is altered, we were interested to clarify this question in detail. Here, we show, using the cecal ligation and puncture (CLP) mouse model of polymicrobial sepsis, that predominantly immature thymocytes are reduced. The number of immature single positive thymocytes was most marked diminished (CLP: 6.54 × 10 ±â€Š3.79 × 10 vs. sham: 4.54 × 10 ±â€Š7.66 × 10 cells/thymus [24 h], CLP: 2.60 × 10 ±â€Š2.14 × 10 vs. sham: 2.17 × 10 ±â€Š1.90 × 10 cells/thymus [48 h]), and was consequently associated with the highest rate of apoptosis (8.4 [CLP] vs. 2.2% [sham]), the reduction in double positive thymocytes being associated with a smaller apoptotic response (number, CLP: 2.33 × 10 ±â€Š1.38 × 10 vs. sham: 1.07 × 10 ±â€Š2.72 × 10 cells/thymus [24 h], CLP: 2.34 × 10 ±â€Š9.08 × 10 vs. sham: 3.5 × 10 ±â€Š9.62 × 10 cells/thymus [48 h]; apoptosis: 2.5% [CLP] vs. 0.7% [sham]). Analysis of T-cell receptor excision circles revealed that the emigration of mature thymocytes was not inhibited. Real-time qPCR analysis revealed upregulation of pro-apoptotic Bim expression and suggested interference between Notch receptor expression on thymocytes and the respective ligands on thymic stromal cells during CLP-dependent sepsis, which might be responsible for the altered thymocyte viability in CLP-dependent sepsis.

Identification and characterisation of a prototype for a new class of competitive PPARγ antagonists.

Understanding of the physiological role of peroxisome proliferator-activated receptor gamma (PPARγ) offers new opportunities for the treatment of cancers, immune disorders and inflammatory diseases. In contrast to PPARγ agonists, few PPARγ antagonists have been studied, though they do exert immunomodulatory effects. Currently, no therapeutically useful PPARγ antagonist is commercially available. The aim of this study was to identify and kinetically characterise a new competitive PPARγ antagonist for therapeutic use. A PPARγ-dependent transactivation assay was used to kinetically characterise (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl)benzyl)oxy)-benzylidene)-hexanoic acid (MTTB) in kidney, T and monocytic cell lines. Cytotoxic effects were analysed and intracellular accumulation of MTTB was assessed by tandem mass spectrometry (LC-MS/MS). Potential interactions of MTTB with the PPARγ protein were suggested by molecular docking analysis. In contrast to non-competitive, irreversible inhibition caused by 2-chloro-5-nitrobenzanilide (GW9662), MTTB exhibited competitive antagonism against rosiglitazone in HEK293T and Jurkat T cells, with IC50 values in HEK293T cells of 4.3µM and 1.6µM, using the PPARγ ligand binding domain (PPARγ-LBD) and the full PPARγ protein, respectively. In all cell lines used, however, MTTB showed much higher intracellular accumulation than GW9662. MTTB alone exhibited weak partial agonistic effects and low cytotoxicity. Molecular docking of MTTB with the PPARγ-LBD supported direct interaction with the nuclear receptor. MTTB is a promising prototype for a new class of competitive PPARγ antagonists. It has weak partial agonistic and clear competitive antagonistic characteristics associated with rapid cellular uptake. Compared to commercially available PPARγ modulators, this offers the possibility of dose regulation of PPARγ and immune responses.

Loss of Nrf2 in bone marrow-derived macrophages impairs antigen-driven CD8(+) T cell function by limiting GSH and Cys availability.

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, ß-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.

Activation of the peroxisome proliferator-activated receptor γ counteracts sepsis-induced T cell cytotoxicity toward alloantigenic target cells.

UNLABELLED: Sepsis still emerges as a major cause of patient death in intensive care units. Therefore, new therapeutic approaches are mandatory. Because during sepsis progression cytotoxic T lymphocytes (CTLs) can be activated in an autoimmune fashion contributing to multiorgan damage, it remains unclear whether CTLs are activated toward alloantigenic cells. This is important for patients receiving an immunosuppressive therapy to permit organ transplantation and, thus, known to be at high risk for developing sepsis. Therefore, we analyzed whether sepsis activates CTL toward alloantigenic target cells and whether this can be inhibited by PPARγ activation, known to block T helper cell responses. To mimic septic conditions, CTLs were isolated from cecal ligation and puncture-operated mice. CTL cytotoxicity was analyzed following a direct alloantigenic activation regime or following classical ex vivo splenocyte-driven activation in a cytotoxicity assay. With this readout, we found that CTL derived from septic mice enhanced cytotoxicity toward alloantigenic target cells, which was lowered by in vivo and ex vivo PPARγ activation. With CTL derived from T cell-specific PPARγ knockout mice, PPARγ activation was ineffective, pointing to a PPARγ-dependent mechanism. In vivo and ex vivo PPARγ activation reduced Fas and granzyme B expression in activated CTL. KEY MESSAGE: In the sepsis CLP mouse model, CTLs are activated toward alloantigenic target cells. Sepsis-mediated alloantigenic CTL activation is blocked in vivo by PPARγ activation. PPARγ deletion or antagonization restored rosiglitazone-dependent inhibition of CTL cytotoxicity. PPARγ inhibits the expression of Fas and granzyme B in CTLs.

Autophagy-dependent PELI3 degradation inhibits proinflammatory IL1B expression.

Lipopolysaccharide (LPS)-induced activation of TLR4 (toll-like receptor 4) is followed by a subsequent overwhelming inflammatory response, a hallmark of the first phase of sepsis. Therefore, counteracting excessive innate immunity by autophagy is important to contribute to the termination of inflammation. However, the exact molecular details of this interplay are only poorly understood. Here, we show that PELI3/Pellino3 (pellino E3 ubiquitin protein ligase family member 3), which is an E3 ubiquitin ligase and scaffold protein in TLR4-signaling, is impacted by autophagy in macrophages (MΦ) after LPS stimulation. We noticed an attenuated mRNA expression of proinflammatory Il1b (interleukin 1, ß) in Peli3 knockdown murine MΦ in response to LPS treatment. The autophagy adaptor protein SQSTM1/p62 (sequestosome 1) emerged as a potential PELI3 binding partner in TLR4-signaling. siRNA targeting Sqstm1 and Atg7 (autophagy related 7), pharmacological inhibition of autophagy by wortmannin as well as blocking the lysosomal vacuolar-type H(+)-ATPase by bafilomycin A1 augmented PELI3 protein levels, while inhibition of the proteasome had no effect. Consistently, treatment to induce autophagy by MTOR (mechanistic target of rapamycin (serine/threonine kinase)) inhibition or starvation enhanced PELI3 degradation and reduced proinflammatory Il1b expression. PELI3 was found to be ubiquitinated upon LPS stimulation and point mutation of PELI3-lysine residue 316 (Lys316Arg) attenuated Torin2-dependent degradation of PELI3. Immunofluorescence analysis revealed that PELI3 colocalized with the typical autophagy markers MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 ß) and LAMP2 (lysosomal-associated membrane protein 2). Our observations suggest that autophagy causes PELI3 degradation during TLR4-signaling, thereby impairing the hyperinflammatory phase during sepsis.

SYNCRIP-dependent Nox2 mRNA destabilization impairs ROS formation in M2-polarized macrophages.

AIMS: During sepsis, macrophages are alternatively activated toward an M2-like phenotype on contact with apoptotic cells (ACs) or their secretion products. Simultaneously, NADPH oxidase-dependent reactive oxygen species (ROS) formation is attenuated, thus contributing to immune paralysis. However, the exact mechanism remains elusive. Here, we provide mechanistic insights into diminished mRNA stability of the NADPH oxidase Nox2 on macrophage M2 polarization and therefore reduced ROS formation in sepsis. RESULTS: Murine J774A.1 macrophages were stimulated with conditioned medium (CM) of apoptotic T cells, which reduced Nox2 mRNA and protein expression, consequently decreasing ROS production. An mRNA pulldown approach coupled to mass spectrometry analysis identified the RNA-binding protein SYNCRIP attached to the Nox2 mRNA 3' untranslated region (3'UTR). The binding of SYNCRIP to the 3'UTR of Nox2 mRNA is attenuated after treatment with CM of apoptotic T cells, followed by Nox2 mRNA destabilization. In in vivo models of polymicrobial sepsis such as cecal ligation and puncture, SYNCRIP was strongly downregulated, which was associated with a decreased Nox2 expression in peritoneal macrophages. INNOVATION: Downregulation of SYNCRIP in macrophages after contact to material of ACs destabilized Nox2 mRNA and impaired ROS formation, thereby contributing to an M2 phenotype shift of macrophages in sepsis. CONCLUSION: M2 polarization of macrophages in sepsis results in an attenuated SYNCRIP binding to the 3'UTR of Nox2 mRNA, destabilizing Nox2 mRNA abundance and expression. Consequently, ROS formation needed to fight against recurrent infections is impaired. In conclusion, SYNCRIP-regulated Nox2 mRNA degradation mediates the hypoinflammatory phase of sepsis.

5-Lipoxygenase contributes to PPARγ activation in macrophages in response to apoptotic cells.

Macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells is a contributing hallmark to immune suppression during the late phase of sepsis. Although the peroxisome proliferator-activated receptor γ (PPARγ) supports this macrophage phenotype switch, it remains elusive how apoptotic cells activate PPARγ. Assuming that a molecule causing PPARγ activation in macrophages originates in the cell membrane of apoptotic cells we analyzed lipid rafts from apoptotic, necrotic, and living human Jurkat T cells which showed the presence of 5-lipoxygenase (5-LO) in lipid rafts of apoptotic cells only. Incubating macrophages with lipid rafts of apoptotic, but not necrotic or living cells, induced PPAR responsive element (PPRE)-driven mRuby reporter gene expression in RAW 264.7 macrophages stably transduced with a 4xPPRE containing vector. Experiments with lipid rafts of apoptotic murine EL4 T cells revealed similar results. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated with the 5-LO inhibitor MK-866 prior to induction of apoptosis, which failed to induce mRuby expression. Similar results were obtained with lipid rafts of apoptotic EL4 T cells preexposed to the 5-LO inhibitors zileuton and CJ-13610. Interestingly, Jurkat T cells overexpressing 5-LO failed to activate PPARγ in macrophages, while their 5-LO overexpressing apoptotic counterparts did. Our results suggest that during apoptosis 5-LO gets associated with lipid rafts and synthesizes ligands that in turn stimulate PPARγ in macrophages.

PPARγ stabilizes HO-1 mRNA in monocytes/macrophages which affects IFN-ß expression.

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-ß 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-ß 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-ß.