NLRC5 Functions beyond MHC I Regulation—What Do We Know So Far?

NLRC5 is a member of the NLR family that acts as a transcriptional activator of MHC class I genes. In line with the function of several related NLR proteins in innate immune responses, there is, however, also ample evidence that NLRC5 contributes to innate and adaptive immune responses beyond the regulation of MHC class I genes. In human and murine cells, for example, NLRC5 was proposed to contribute to inflammatory and type I interferon responses. The role of NLRC5 in these and other cellular processes is hitherto still not well understood and blurred by discrepancies in the reported data. Here, we provide a detailed and critical discussion of the available experimental data on the emerging biological functions of NLRC5 in innate immune responses in men and mice. Better awareness of the multiple roles of NLRC5 will help to define its overall contribution to immune responses and cancer

The H2S Donor Sodium Thiosulfate (Na2S2O3) Does Not Improve Inflammation and Organ Damage After Hemorrhagic Shock in Cardiovascular Healthy Swine

We previously demonstrated marked lung-protective properties of the H2S donor sodium thiosulfate (Na2S2O3, STS) in a blinded, randomized, controlled, long-term, resuscitated porcine model of swine with coronary artery disease, i.e., with decreased expression of the H2S-producing enzyme cystathionine-γ-lyase (CSE). We confirmed these beneficial effects of STS by attenuation of lung, liver and kidney injury in mice with genetic CSE deletion (CSE-ko) undergoing trauma-and-hemorrhage and subsequent intensive care-based resuscitation. However, we had previously also shown that any possible efficacy of a therapeutic intervention in shock states depends both on the severity of shock as well as on the presence or absence of chronic underlying co-morbidity. Therefore, this prospective, randomized, controlled, blinded experimental study investigated the effects of the STS in cardiovascular healthy swine. After anesthesia and surgical instrumentation, 17 adult Bretoncelles-Meishan-Willebrand pigs were subjected to 3 hours of hemorrhage by removal of 30% of the blood volume and titration of the mean arterial pressure (MAP) ≈ 40 ± 5 mmHg. Afterwards, the animals received standardized resuscitation including re-transfusion of shed blood, fluids, and, if needed, continuous i.v. noradrenaline to maintain MAP at pre-shock values. Animals were randomly allocated to either receive Na2S2O3 or vehicle control starting 2 hours after initiation of shock until 24 hours of resuscitation. The administration of Na2S2O3 did not alter survival during the observation period of 68 hours after the initiation of shock. No differences in cardio-circulatory functions were noted despite a significantly higher cardiac output, which coincided with significantly more pronounced lactic acidosis at 24 hours of resuscitation in the Na2S2O3 group. Parameters of liver, lung, and kidney function and injury were similar in both groups. However, urine output was significantly higher in the Na2S2O3 group at 24 hours of treatment. Taken together, this study reports no beneficial effect of Na2S2O3 in a clinically relevant model of hemorrhagic shock-and-resuscitation in animals without underlying chronic cardiovascular co-morbidity

The effect of sodium thiosulfate on immune cell metabolism during porcine hemorrhage and resuscitation

Introduction Sodium thiosulfate (Na2S2O3), an H2S releasing agent, was shown to be organ-protective in experimental hemorrhage. Systemic inflammation activates immune cells, which in turn show cell type-specific metabolic plasticity with modifications of mitochondrial respiratory activity. Since H2S can dose-dependently stimulate or inhibit mitochondrial respiration, we investigated the effect of Na2S2O3 on immune cell metabolism in a blinded, randomized, controlled, long-term, porcine model of hemorrhage and resuscitation. For this purpose, we developed a Bayesian sampling-based model for 13C isotope metabolic flux analysis (MFA) utilizing 1,2-13C2-labeled glucose, 13C6-labeled glucose, and 13C5-labeled glutamine tracers. Methods After 3 h of hemorrhage, anesthetized and surgically instrumented swine underwent resuscitation up to a maximum of 68 h. At 2 h of shock, animals randomly received vehicle or Na2S2O3 (25 mg/kg/h for 2 h, thereafter 100 mg/kg/h until 24 h after shock). At three time points (prior to shock, 24 h post shock and 64 h post shock) peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from whole blood, and cells were investigated regarding mitochondrial oxygen consumption (high resolution respirometry), reactive oxygen species production (electron spin resonance) and fluxes within the metabolic network (stable isotope-based MFA). Results PBMCs showed significantly higher mitochondrial O2 uptake and lower O 2 • − production in comparison to granulocytes. We found that in response to Na2S2O3 administration, PBMCs but not granulocytes had an increased mitochondrial oxygen consumption combined with a transient reduction of the citrate synthase flux and an increase of acetyl-CoA channeled into other compartments, e.g., for lipid biogenesis. Conclusion In a porcine model of hemorrhage and resuscitation, Na2S2O3 administration led to increased mitochondrial oxygen consumption combined with stimulation of lipid biogenesis in PBMCs. In contrast, granulocytes remained unaffected. Granulocytes, on the other hand, remained unaffected. O 2 • − concentration in whole blood remained constant during shock and resuscitation, indicating a sufficient anti-oxidative capacity. Overall, our MFA model seems to be is a promising approach for investigating immunometabolism; especially when combined with complementary methods

Die Effekte von Natriumthiosulfat bei der Akuttherapie von Trauma und Hämorrhagie in Cystathionin-γ-Lyase-knockout Mäusen mit vorbestehender COPD

Traumatische Thoraxverletzungen weisen bei polytraumatisierten Patienten eine Letalität von ca. 25 % auf. Hierbei ist der hämorrhagische Schock (HS) eine bedeutende Komplikation und für etwa 30 -40 % der traumabedingten Todesfälle verantwortlich. Schwere Komplikationen des Thoraxtraumas mit HS umfassen die akute Lungenverletzung (ALI), das akute Nierenversagen (AKI) und multiples Organversagen (MOF). Bei diesen Traumafolgen ist die überschießende systemische Entzündungsreaktion ein zentrales Element der posttraumatischen Morbiditätsausprägung. Diese entwickelt sich unter anderem als Reaktion auf die traumabedingte Gewebeschädigung mit darauffolgender Gewebehypoxie. Ebenfalls durch eine chronische systemische Entzündungsreaktion, sowie einer alveolären Hypoxie mit darauffolgender Hypoxämie gekennzeichnet ist die chronisch obstruktive Lungenerkrankung, die COPD. Hierbei handelt es sich um eine häufige Erkrankung der kleinen Atemwege mit extrapulmonalen Auswirkungen, hauptsächlich verursacht durch das Inhalieren von Zigarettenrauch (90 %). Die Kombination von COPD, Thoraxtrauma und HS verstärkt die posttraumatische Entzündungsreaktion signifikant. Als zentrales Element für Therapieansätze einer solchen Traumakonstellation gilt deshalb die Modulation der überschießenden posttraumatischen Entzündungsreaktion. Dem Gasotransmitter Schwefelwasserstoff (H2S) werden in Bezug auf die Behandlung von Trauma und Hämorrhagie in der Literatur sowohl positive als auch negative Eigenschaften zugeschrieben. Die Steigerung der H2S-Verfügbarkeit zeigte in Vorversuchen in verschiedenen Schockmodellen jedoch eine protektive Wirkung. Als Ausgangspunkt in dieser Studie diente H2S als Gasotransmitter mit seinen charakteristischen Eigenschaften wie der Regulation des Gefäßtonus, seiner antioxidativen Wirkung und seinen antiinflammatorischen Eigenschaften. Die exogene H2S-Supplementierung hatte mit den bisher genutzten Substanzen aufgrund von Nebenwirkungen keinen therapeutischen Erfolg. Die vorliegende Studie verwendete ein Mausmodell mit stabilem Knock-out der Cystathionin-γ-Lyase (CSE), einem zentralen Enzym der endogenen H2S-Produktion. Dabei wurden die Effekte einer Natriumthiosulfat (NTS; Na2S2O3) vermittelten H2S-Supplementation am Modell des stumpfen Thoraxtraumas bei vorbestehender COPD untersucht. Hierzu wurden zwei Behandlungsgruppen gebildet, von denen eine mit NTS behandelt wurde und die andere mit einer Kontrollsubstanz (Vehicel). Nach dem Traumaereignis erfolgte die Hämorrhagie mit anschließender Reperfusion und die Behandlung der beiden Gruppen entsprechend ihrer Zuteilung. Es folgte dann eine 6-stündige intensivmedizinische Überwachungsphase, bevor das Experiment beendet wurde. Die physiologischen und intensivmedizinischen Daten der Versuchstiere beider Behandlungsgruppen wurden anschließend biometrisch verglichen, um einen therapeutischen Effekt von Natriumthiosulfat im verwendeten Versuchsmodell zu untersuchen. Als Primärkriterien für die Beurteilung eines positiven therapeutischen Effekts wurden ein verringerter Noradrenalinverbrauch, sowie eine verbesserte Kreatinin-Clearance festgelegt. Dabei zeigten sich keine signifikanten Hinweise auf eine Wirkung der Therapie mit Natriumthiosulfat im vorliegenden Modellversuch. Diese Ergebnisse bestätigten die Eignung von Natriumthiosulfat als Medikament mit günstigem Nebenwirkungsprofil in der Behandlung von Trauma und Hämorrhagie am vorliegenden Modellorganismus. Betrachtet man die Ergebnisse dieser Arbeit im Kontext von vorherigen Studien mit ähnlichem Versuchsaufbau und ebenfalls unter Verwendung von CSE-Knockout-Mäusen einmal ohne- und dann mit Typ I Diabetes mellitus als Begleiterkrankung, so zeichnet sich ein eher heterogenes Bild. Ohne zusätzliche Begleiterkrankungen wies die Behandlung mit Natriumthiosulfat positive Wirkungen auf die Organe, sowie entzündungshemmende Eigenschaften auf. Im Gegensatz dazu zeigte Natriumthiosulfat an Versuchstieren, die zusätzlich an Diabetes mellitus Typ 1 litten, keine therapeutische Wirkung. In der Zusammenschau deuten diese Ergebnisse darauf hin, dass der Faktor Komorbidität einen entscheidenden Einfluss auf die Ausprägung der Wirkung von Natriumthiosulfat im hier untersuchten Traumamodell hat

Control of Morphological Differentiation of Streptomyces coelicolor A3(2) by Phosphorylation of MreC and PBP2

During morphological differentiation of Streptomyces coelicolor A3(2), the sporogenic aerial hyphae are transformed into a chain of more than fifty spores in a highly coordinated manner. Synthesis of the thickened spore envelope is directed by the Streptomyces spore wall synthesizing complex SSSC which resembles the elongasome of rod-shaped bacteria. The SSSC includes the eukaryotic type serine/threonine protein kinase (eSTPK) PkaI, encoded within a cluster of five independently transcribed eSTPK genes (SCO4775-4779). To understand the role of PkaI in spore wall synthesis, we screened a S. coelicolor genomic library for PkaI interaction partners by bacterial two-hybrid analyses and identified several proteins with a documented role in sporulation. We inactivated pkaI and deleted the complete SCO4775-4779 cluster. Deletion of pkaI alone delayed sporulation and produced some aberrant spores. The five-fold mutant NLΔ4775-4779 had a more severe defect and produced 18% aberrant spores affected in the integrity of the spore envelope. Moreover, overbalancing phosphorylation activity by expressing a second copy of any of these kinases caused a similar defect. Following co-expression of pkaI with either mreC or pbp2 in E. coli, phosphorylation of MreC and PBP2 was demonstrated and multiple phosphosites were identified by LC-MS/MS. Our data suggest that elaborate protein phosphorylation controls activity of the SSSC to ensure proper sporulation by suppressing premature cross-wall synthesis

Effect of overbalancing phosphorylation activity on the integrity of spore envelopes.

<p>Live-dead staining of spore chains of the eSTPK mutants NLΔPkaI and NLΔ4775–4779 (A) revealed the presence of dead spores (red) or spores without DNA (black). In contrast, spore chains of the parental M145 strain (A) only contained viable spores (green). Expression of a second copy of any eSTPK gene of cluster <i>SCO4775-4779</i> (B-F) caused a similar sporulation defect in <i>S</i>. <i>coelicolor</i> M145, NLΔPkaI, or NLΔ4775–4779. None of the eSTPK genes was able to complement aberrant sporulation of the five-fold mutant NLΔ4775–4779. A, no plasmid integrated; B, :: pSET152-pkaH; C, :: pSET152-SCO4776; D, :: pSET152-pkaD; E, :: pSET152-pkaI; F, :: pSET152-pkaJ. Bar = 5 μm.</p

Effect of Ser/Thr kinases on the viability of spores (A) and the resistance of germinating spores to vancomycin (B).

<p>Spore chains were stained with the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes) and observed by fluorescence microscopy. Percentage of viable (green), dead (red) and spores without DNA (black) is given for each strain. Spores of the different strains were plated onto LB agar and filter discs containing 5 μg vancomycin were applied. Whereas, M145 and the <i>pkaI</i> mutant NLΔPkaI were resistant, NLΔ4775–4779 spores showed vancomycin sensitivity, suggesting an impaired spore wall. Vancomycin sensitivity of M145 or NLΔPkaI was also caused by expressing a second copy of each kinase gene, with the exception of <i>pkaI</i>. Supplementation of the agar plates with 3 mM MgCl_2, known to rescue mutants impaired in cell wall synthesis restored vancomycin resistance to all strains. A, no plasmid integrated; B, :: pSET152-pkaH; C, :: pSET152-SCO4776; D, :: pSET152-pkaD; E, :: pSET152-pkaI; F, :: pSET152-pkaJ.</p

Effect of overbalancing phosphorylation activity on proper sporulation of <i>S</i>. <i>coelicolor</i>.

<p>Phase contrast microscopy of spore chains revealed the presence of aberrant spores (arrows) in eSTPK mutants NLΔPkaI and NLΔ4775–4779 (A). In contrast, spore chains of the parental M145 strain (A) and the complemented mutant NLΔPkaI::pSET-pkaI (E) contain mainly regular ovoid spores. Not only deletion, but also expression of a second copy of any eSTPK gene of cluster <i>SCO4775-4779</i> causes a similar sporulation defect (B-F, white arrows) in <i>S</i>. <i>coelicolor</i> M145, NLΔPkaI, or NLΔ4775–4779. None of the eSTPK genes is able to complement aberrant sporulation of the five-fold mutant NLΔ4775–4779. A, no plasmid integrated; B, :: pSET152-pkaH; C, :: pSET152-SCO4776; D, :: pSET152-pkaD; E, :: pSET152-pkaI; F, :: pSET152-pkaJ. Bar = 5 μm.</p

Phosphorylation of PBP2 by PkaI.

<p><i>S</i>. <i>coelicolor pbp2</i> with a S-tag encoding sequence was co-expressed with <i>pkaI</i> in <i>E</i>. <i>coli</i>. PBP2_S-tag was purified under denaturing conditions by affinity chromatography. PkaI with an N-terminal His-tag was purified by Ni-NTA chromatography under native conditions. Purified His_PkaI and PBP2_S-tag proteins (bold letters and underlining indicate, which protein was purified) were separated on an SDS polyacrylamide gel and stained with Coomassie blue (<b>A</b>). Phosphorylated proteins were identified by ProQ Diamond staining. The white arrow indicates auto-phosphorylated His_PkaI, while the black arrow marks phosphorylated PBP2_S-tag. Immunoblotting with Anti-S-tag antibodies confirmed the identity of PBP2_S-tag. Domain architecture of PBP2 and positions of the most likely phosphosites (<b>B</b>). Predicted Pfam domains (PBP dimerization, dark grey, PBP transpeptidase, light grey), a transmembrane helix (TM) and the positions of phosphorylated S/T residues, identified by LC-MS/MS, are indicated.</p