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In search for effective multi-targeting drug ligands (MTDLs) to address low-grade inflammatory changes of metabolic disorders, we rationally designed some novel glitazones-like compounds. This was achieved by incorporating prominent pharmacophoric motifs from previously reported COX-2, 15-LOX and PPARγ ligands. Challenging our design with pre-synthetic docking experiments on PPARγ showed encouraging results. In vitro tests have identified 4 compounds as simultaneous partial PPARγ agonist, potent COX-2 antagonist (nanomolar IC50 values) and moderate 15-LOX inhibitor (micromolar IC50 values). We envisioned such outcome as a prototypical balanced modulation of the 3 inflammatory targets. In vitro glucose uptake assay defined six compounds as insulin-sensitive and the other two as insulin-independent glucose uptake enhancers. Also, they were able to induce PPARγ nuclear translocation in immunohistochemical analysis. Their anti-inflammatory potential has been translated to effective inhibition of monocyte to macrophage differentiation, suppression of LPS-induced inflammatory cytokine production in macrophages, as well as significant in vivo anti-inflammatory activity. Ligand co-crystallized PPARγ X-ray of one of MTDLs has identified new clues that could serve as structural basis for its partial agonism. Docking of the most active compounds into COX-2 and 15-LOX active sites, pinpointed favorable binding patterns, similar to those of the co-crystallized ligands. Finally, in silico assessment of pharmacokinetics, physicochemical properties, drug-likeness and ligand efficiency indices was performed. Hence, we anticipate that the prominent biological profile of such series will rationalize relevant anti-inflammatory drug development endeavors.

Animal welfare and the refinement of experimental procedures are fundamental aspects of biomedical research. They provide the basis for robust experimental designs and reproducibility of results. In many countries, the determination of welfare is a mandatory legal requirement and implies the assessment of the degree of the severity that an animal experiences during an experiment. However, for an effective severity assessment, an objective and exact approach/system/strategy is needed. In light of these demands, we have developed the Relative Severity Assessment (RELSA) score.This comprehensive composite score was established on the basis of physiological and behavioral data from a surgical mouse study. Body weight, the Mouse Grimace Scale score, burrowing behavior, and the telemetry-derived parameters heart rate, heart rate variability, temperature, and general activity were used to investigate the quality of indicating severity during postoperative recovery. The RELSA scores not only revealed individual severity levels but also allowed a comparison of severity in distinct mouse models addressing colitis, sepsis, and restraint stress using a k-means clustering approach with the maximum achieved RELSA scores.We discriminated and classified data from sepsis nonsurvivors into the highest relative severity level. Data from mice after intraperitoneal transmitter implantation and sepsis survivor al were located in the next lower cluster, while data from mice subjected to colitis and restraint stress were placed in the lowest severity cluster. Analysis of individual variables and their combinations revealed model- and time-dependent contributions to severity levels.In conclusion, we propose the RELSA score as a validated tool for objective real-time applicability in severity assessment and as a first step towards a unified and accessible risk assessment tool in biomedical research. As an effective severity assessment system, it will fundamentally improve animal welfare, as well as data quality and reproducibility.

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.

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 x 10(4) +/- 3.79 x 10(4) vs. sham: 4.54 x 10(5) +/- 7.66 x 10(4) cells/thymus [24 h], CLP: 2.60 x 10(2) +/- 2.14 x 10(2) vs. sham: 2.17 x 10(5) +/- 1.90 x 10(4) 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 x 10(6) +/- 1.38 x 10(6) vs. sham: 1.07 x 10(7) +/- 2.72 x 10(6) cells/thymus [24 h], CLP: 2.34 x 10(3) +/- 9.08 x 10(2) vs. sham: 3.5 x 10 6 +/- 9.62 x 10(5) 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.

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.

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.

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.

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.

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 PPAR 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 f ound that CTL derived from septic mice enhanced cytotoxicity toward alloantigenic target cells, which was lowered by in vivo and ex vivo PPAR activation. With CTL derived from T cell-specific PPAR knockout mice, PPAR activation was ineffective, pointing to a PPAR-dependent mechanism. In vivo and ex vivo PPAR 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 PPAR activation.PPAR deletion or antagonization restored rosiglitazone-dependent inhibition of CTL cytotoxicity.PPAR inhibits the expression of Fas and granzyme B in CTLs.

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.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.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.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.

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.

Translating murine data to the human situation, we proposed that the level of peroxisome proliferator-activated receptor g (PPARg) expression in T cells from septic patients correlates with clinical outcome. In this preliminary report, we analyzed PPARg mRNA expression in CD3+ T cells derived from blood of a very small number of septic patients (n = 18) on various days up to 2 weeks after the initial diagnosis. CD3+ T cell count was determined by flow cytometry. T cells from n = 11 healthy donors were included as controls. Maximal PPARg mRNA expression was observed on the day of sepsis diagnosis (day 0; 5,896 ± 1,523 copies PPARg mRNA/25 ng mRNA, *P

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, β) 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 β) 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.

Introduction Sepsis originates from an uncontrolled inflammatory response. Despite intensive research, sepsis remains a major cause of death in ICUs. Therefore, new therapeutic approaches are mandatory. Taking into account that during sepsis progression cytotoxic T cells (CTL) are activated in an autoimmune fashion contributing to multiorgan damage, it remains unclear whether CTL are activated towards alloantigenic cells as well. This is especially important for patients receiving an immune suppressive therapy to permit organ transplantation and thus known to be at high risk for developing sepsis. Therefore, we analyzed whether sepsis activates CTL towards alloantigenic target cells and whether this can be inhibited by PPARg activation, known to block T-helper cell responses. Methods To characterize whether sepsis activates CTL and whether this can be inhibited by PPARg activation, we used an ex vivo cytotoxicity assay to analyze CD8+ T-cell-dependent cytotoxicity. Responder CD8+ T cells were isolated from C57Bl/6N PPARg wildtype (PPARgfl/fl) and T-cell specific knockout (Tc-PPARg-/-) mice (haplotype H2Kb) following cecal ligation and puncture (CLP) versus sham treatment. P815 mastocytoma cells, a cell line originally derived from DBA/2 mice (haplotype H2Kd), were used as alloantigenic target cells. Pharmacological inhibition and/or activation of PPARg in vivo and ex vivo was performed to clarify the impact of PPARg in blocking CTL-dependent cytotoxicity. In vivo, PPARg activity in wildtype mice was pharmacologically inhibited by the irreversible antagonist GW9662 or induced by the thiazolidinedione rosiglitazone. Systemic application of both compounds was performed intraperitoneally. A classic splenocyte-driven stimulation protocol to activate CTL was carried out as control. Results CTL isolated from septic mice showed enhanced cytotoxicity towards alloantigenic P815 target cells. Enhanced cytotoxicity was effectively reduced by both PPARg activation in vivo and ex vivo. In line, in CTL isolated from T-cell-specific PPARg knockout (Tc-PPARg-/-) mice PPARg activation was ineffective, strengthening a PPARg-dependent mechanism. At the molecular level in vivo and ex vivo activation of PPARg reduced Fas and granzyme B expression in activated CTL, which might explain reduced cytotoxicity. Conclusion Our study therefore suggests PPARg activation in vivo to attenuate CTL-dependent alloantigenic cytotoxicity to possibly inhibit acute organ rejection.

Background: The multicomponent phagocytic NADPH oxidase produces reactive oxygen species (ROS) after activation by microorganisms or inflammatory mediators. In the hypoinflammatory phase of sepsis, macrophages are alternatively activated by contact with apoptotic cells or their secretion products. This inhibits NADPH oxidase and leads to attenuated ROS production and furthermore contributes among others to a hyporeactive host defense. Due to this immune paralysis, sepsis patients suffer from recurrent and secondary infections. We focused on the catalytic subunit of NADPH oxidase, the transmembrane protein NOX2. We assume that after induction of sepsis the expression of NOX2 is reduced and hence ROS production is decreased. Methods: We induced polymicrobial sepsis in mice by cecal ligation and puncture. The ability of peritoneal macrophages (PMs) to produce ROS was determined by FACS via hydroethidine assay. NOX2 expression of PMs was determined by western blot and qPCR. To elucidate the mechanism causing mRNA destabilization, we performed in vitro experiments using J774 macrophages. To obtain an alternatively activated phenotype, macrophages were stimulated with conditioned medium from apoptotic T cells (CM). By luciferase assays we figured out a 3'UTR-dependent regulation of NOX2 mRNA stability. Assuming that a protein is involved in the mRNA degradation, we performed a RNA pulldown with biotinylated NOX2-3'UTR constructs followed by mass spectrometry. We verified the role of SYNCRIP by siRNA approach. Additionally, we overexpressed NOX2 in J774 cells and analyzed the ROS production (w/wo CM treatment) by FACS. Results: We found an impaired expression of NOX2 at RNA and protein level along with decreased ROS production after induction of sepsis in mice as well as stimulating J774 macrophages with CM of apoptotic T cells. This is due to a time-dependent NOX2 mRNA degradation depending on SYNCRIP, a RNA-binding protein, which stabilizes NOX2 mRNA through binding to its 3'UTR under normal conditions. In line, knockdown of SYNCRIP also decreases NOX2 mRNA expression. We assume that a CM-dependent modification or degradation of SYNCRIP prevents its stabilizing function. As the overexpression of NOX2 restores ROS production of CM-treated J774 cells, we assume that NOX2 expression is crucial for maintaining NADPH activity during the hypoinflammatory phase of sepsis. Conclusion: Our data imply a regulatory impact of SYNCRIP on NOX2 stability during the late phase of sepsis. Therefore, further understanding of the regulation of NADPH oxidase could lead to the design of a therapy to reconstitute NADPH oxidase function, finally improving immune function in sepsis patients.

Background: In macrophages Toll-like receptor 4 (TLR4) is activated in response to lipopolysaccharide (LPS) and induces proinflammatory cytokine expression. Therefore, mechanisms terminating proinflammatory gene expression are important. Autophagy plays a central role in controlling innate immune responses by lysosomal degradation of signaling proteins, thus contributing to the resolution of inflammation. Autophagic proteins like p62 directly interact with molecules involved in the TLR4-signaling pathway, but a correlation with the IRAK E3 ligase and scaffold protein Pellino3 remains obscure. Hence, we are interested in elucidating the function of Pellino3 to prove our hypothesis that it is a key regulator in the TLR4-signaling cascade. Methods: We used the cecal ligation and puncture (CLP) mouse model causing polymicrobial sepsis to analyze Pellino3 protein and mRNA expression. Furthermore, we induced endotoxemia in RAW264.7 mouse macrophages by LPS treatment to verify in vivo experiments. Lentiviral Pellino3 knockdown in RAW264.7 macrophages was used for cytokine measurements at mRNA level. To analyze potential Pellino3 binding partners in TLR4-signaling by mass spectrometry (MS), we overexpressed FLAG-tagged Pellino3 in RAW264.7 macrophages, treated cells for 3, 6 and 24 hours with LPS and immunoprecipitated Pellino3 via its FLAG-tag. To consider Pellino3 degradation as a result of p62-mediated autophagy, we transiently knocked down p62 by siRNA in RAW264.7 macrophages and also pharmacologically blocked LPS-induced autophagy by Bafilomycin A1. Results: We demonstrated Pellino3 protein degradation in primary CD11b+ splenocytes after 24 hours following CLP operation and confirmed this in RAW264.7 macrophages after 24-hour LPS stimulation. Knockdown of Pellino3 attenuates proinflammatory cytokines, for example IL-6 mRNA, after 6 hours of LPS. Furthermore, we found by MS and verifying immunoprecipitation experiments that p62 is a Pellino3 binding partner, thus targeting Pellino3 for degradation. In line, both p62 knockdown and Bafilomycin A1 treatment prevent Pellino3 degradation, supporting an autophagic mechanism. Conclusion: Our observations highlight a regulatory role of Pellino3 on TLR4 signaling. Thus, antagonism of Pellino3 in the hyperinflammatory phase of sepsis may counteract the cytokine storm. Furthermore, stabilization of Pellino3 by inhibition of autophagy in the hypoinflammatory phase of sepsis may improve immunity. In consideration of these two conflictive sepsis phases, modulation of Pellino3 may provide a new strategy for the development of a therapy approach in sepsis.

Introduction T-cell depletion is a marker of the hypo-inflammatory phase of sepsis [1,2]. Systemic T-cell loss could be caused i.a. by thymus involution during sepsis, thus contributing to immune paralysis [3]. Usually, thymic T-cell egress is mediated by a sphingosine-1-phosphate (S1P) gradient. That is, T cells leave the thymus towards an increased S1P-level in the periphery (blood/lymph) [4]. A disruption of this S1P gradient blocks T-cell emigration. S1P is produced by sphingosine kinases 1 and 2 (SPHK1 and SPHK2), which are ubiquitously expressed [5]. Apoptotic cells are known to release S1P [6]. We suggest that apoptotic events in the thymus during sepsis increase S1P levels, thereby disrupting the gradient and preventing egress of T cells from the thymus. Methods We used a murine polymicrobial sepsis model to analyze thymus involution. Sepsis is induced in wildtype mice, SPHK1-/-, or SPHK2-/- mice by cecal ligation and puncture (CLP). Thymus involution was determined by analyzing the T-cell amount of single-positive mature (CD4+CD8- vs. CD4-CD8+), double-positive late immature (CD4+/CD8+), and double-negative early immature cells by FACS analysis. T cells from SPHK1-/- or SPHK2-/- mice, which produce less S1P, and wildtype mice treated with a SPHK1 inhibitor were used to characterize whether T cells in these mice have a higher rate of emigration compared to control mice. Thymic and serum S1P levels were quantified by LC-MS/MS. Apoptosis was determined by annexin V FACS staining. SPHK mRNA levels were determined by qPCR. Results The thymus of septic mice showed more CD3-positive cells but a decreased number of CD4/CD8 double-positive T cells, pointing to a thymic retention of single-positive mature T cells. In line with our assumption, CLP causes apoptosis of thymocytes and increases SPHK1 mRNA expression. Concomitantly S1P levels are increased in the thymus and consequently decreased in serum following CLP. The knockout of the S1P producing sphingosine-kinases SPHK1 or SPHK2 and the pharmacological inhibition of SPHK1 restores T-cell egress as indicated by an increase of double-positive immature T cells compared to wildtype mice. Conclusion Our data suggest that inhibition of SPHK1-mediated S1P generation during sepsis restores thymic T-cell egress, which might improve septic outcome. Therefore, understanding mechanisms of thymus involution during sepsis may help to reconstitute thymic function, finally improving immune reactions in the hypoinflammatory phase of sepsis.