Gut microbiome-derived glycine lipids are diet-dependent modulators of hepatic injury and atherosclerosis.

Oral and gut Bacteroidetes produce unique classes of serine-glycine lipodipeptides and glycine aminolipids that signal through host Toll-like receptor 2. These glycine lipids have also been detected in human arteries, but their effects on atherosclerosis are unknown. Here, we sought to investigate the bioactivity of bacterial glycine lipids in mouse models of atherosclerosis. Lipid 654 (L654), a serine-glycine lipodipeptide species, was first tested in a high-fat diet (HFD)-fed Ldlr-/- model of atherosclerosis. Intraperitoneal administration of L654 over 7 weeks to HFD-fed Ldlr-/- mice resulted in hypocholesterolemic effects and significantly attenuated the progression of atherosclerosis. We found that L654 also reduced liver inflammatory and extracellular matrix gene expression, which may be related to inhibition of macrophage activation as demonstrated in vivo by lower major histocompatibility complex class II gene expression and confirmed in cell experiments. In addition, L654 and other bacterial glycine lipids in feces, liver, and serum were markedly reduced alongside changes in Bacteroidetes relative abundance in HFD-fed mice. Finally, we tested the bioactivities of L654 and related lipid 567 in chow-fed Apoe-/- mice, which displayed much higher fecal glycine lipids relative to HFD-fed Ldlr-/- mice. Administration of L654 or lipid 567 for 7 weeks to these mice reduced the liver injury marker alanine aminotransferase, but other effects seen in Ldlr-/- were not observed. Therefore, we conclude that conditions in which gut microbiome-derived glycine lipids are lost, such as HFD, may exacerbate the development of atherosclerosis and liver injury, whereas correction of such depletion may protect from these disorders.

Frequency-Dependent Properties of the Hyperpolarization-Activated Cation Current, If, in Adult Mouse Heart Primary Pacemaker Myocytes.

A number of distinct electrophysiological mechanisms that modulate the myogenic spontaneous pacemaker activity in the sinoatrial node (SAN) of the mammalian heart have been investigated extensively. There is agreement that several (3 or 4) different transmembrane ionic current changes (referred to as the voltage clock) are involved; and that the resulting net current interacts with direct and indirect effects of changes in intracellular Ca2+ (the calcium clock). However, significant uncertainties, and important knowledge gaps, remain concerning the functional roles in SAN spontaneous pacing of many of the individual ion channel- or exchanger-mediated transmembrane current changes. We report results from patch clamp studies and mathematical modeling of the hyperpolarization-activated current, If, in the generation/modulation of the diastolic depolarization, or pacemaker potential, produced by individual myocytes that were enzymatically isolated from the adult mouse sinoatrial node (SAN). Amphotericin-mediated patch microelectrode recordings at 35 °C were made under control conditions and in the presence of 5 or 10 nM isoproterenol (ISO). These sets of results were complemented and integrated with mathematical modeling of the current changes that take place in the range of membrane potentials (-70 to -50 mV), which corresponds to the 'pacemaker depolarization' in the adult mouse SAN. Our results reveal a very small, but functionally important, approximately steady-state or time-independent current generated by residual activation of If channels that are expressed in these pacemaker myocytes. Recordings of the pacemaker depolarization and action potential, combined with measurements of changes in If, and the well-known increases in the L-type Ca2+ current, ICaL, demonstrated that ICaL activation, is essential for myogenic pacing. Moreover, after being enhanced (approximately 3-fold) by 5 or 10 nM ISO, ICaL contributes significantly to the positive chronotropic effect. Our mathematical model has been developed in an attempt to better understand the underlying mechanisms for the pacemaker depolarization and action potential in adult mouse SAN myocytes. After being updated with our new experimental data describing If, our simulations reveal a novel functional component of If in adult mouse SAN. Computational work carried out with this model also confirms that in the presence of ISO the residual activation of If and opening of ICaL channels combine to generate a net current change during the slow diastolic depolarization phase that is essential for the observed accelerated pacemaking rate of these SAN myocytes.

Physiological Roles of the Rapidly Activated Delayed Rectifier K+ Current in Adult Mouse Heart Primary Pacemaker Activity.

Robust, spontaneous pacemaker activity originating in the sinoatrial node (SAN) of the heart is essential for cardiovascular function. Anatomical, electrophysiological, and molecular methods as well as mathematical modeling approaches have quite thoroughly characterized the transmembrane fluxes of Na+, K+ and Ca2+ that produce SAN action potentials (AP) and 'pacemaker depolarizations' in a number of different in vitro adult mammalian heart preparations. Possible ionic mechanisms that are responsible for SAN primary pacemaker activity are described in terms of: (i) a Ca2+-regulated mechanism based on a requirement for phasic release of Ca2+ from intracellular stores and activation of an inward current-mediated by Na+/Ca2+ exchange; (ii) time- and voltage-dependent activation of Na+ or Ca2+ currents, as well as a cyclic nucleotide-activated current, If; and/or (iii) a combination of (i) and (ii). Electrophysiological studies of single spontaneously active SAN myocytes in both adult mouse and rabbit hearts consistently reveal significant expression of a rapidly activating time- and voltage-dependent K+ current, often denoted IKr, that is selectively expressed in the leading or primary pacemaker region of the adult mouse SAN. The main goal of the present study was to examine by combined experimental and simulation approaches the functional or physiological roles of this K+ current in the pacemaker activity. Our patch clamp data of mouse SAN myocytes on the effects of a pharmacological blocker, E4031, revealed that a rapidly activating K+ current is essential for action potential (AP) repolarization, and its deactivation during the pacemaker potential contributes a small but significant component to the pacemaker depolarization. Mathematical simulations using a murine SAN AP model confirm that well known biophysical properties of a delayed rectifier K+ current can contribute to its role in generating spontaneous myogenic activity.

A novel phosphoglycerol serine-glycine lipodipeptide of Porphyromonas gingivalis is a TLR2 ligand.

Porphyromonas gingivalis is a Gram-negative anaerobic periodontal microorganism strongly associated with tissue-destructive processes in human periodontitis. Following oral infection with P. gingivalis, the periodontal bone loss in mice is reported to require the engagement of Toll-like receptor 2 (TLR2). Serine-glycine lipodipeptide or glycine aminolipid classes of P. gingivalis engage human and mouse TLR2, but a novel lipid class reported here is considerably more potent in engaging TLR2 and the heterodimer receptor TLR2/TLR6. The novel lipid class, termed Lipid 1256, consists of a diacylated phosphoglycerol moiety linked to a serine-glycine lipodipeptide previously termed Lipid 654. Lipid 1256 is approximately 50-fold more potent in engaging TLR2 than the previously reported serine-glycine lipid classes. Lipid 1256 also stimulates cytokine secretory responses from peripheral blood monocytes and is recovered in selected oral and intestinal Bacteroidetes organisms. Therefore, these findings suggest that Lipid 1256 may be a microbial TLR2 ligand relevant to chronic periodontitis in humans.

Glycine Lipids of Porphyromonas gingivalis Are Agonists for Toll-Like Receptor 2.

The serine-glycine dipeptide lipid classes, including lipid 430 and lipid 654, are produced by the periodontal pathogen Porphyromonas gingivalis and can be detected in lipid extracts of diseased periodontal tissues and teeth of humans. Both serine-glycine lipid classes were previously shown to engage human and mouse Toll-like receptor 2 (TLR2) and to inhibit mouse osteoblast differentiation and function through engagement of TLR2. It is not clear if other lipids related to serine-glycine lipids are also produced by P. gingivalis The goal of this investigation was to determine whether P. gingivalis produces additional lipid classes similar to the serine-glycine lipids that possess biological properties. P. gingivalis (ATCC 33277) was grown in broth culture, and lipids were extracted and fractionated by high-performance liquid chromatography (HPLC). Lipids were separated using semipreparative HPLC, and specific lipid classes were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometric approaches. Two glycine lipid classes were identified, termed lipid 567 and lipid 342, and these lipid classes are structurally related to the serine-glycine dipeptide lipids. Both glycine lipid classes were shown to promote TLR2-dependent tumor necrosis factor alpha (TNF-α) release from bone marrow macrophages, and both were shown to activate human embryonic kidney (HEK) cells through TLR2 and TLR6 but not TLR1. These results demonstrate that P. gingivalis synthesizes glycine lipids and that these lipids engage TLR2 similarly to the previously reported serine-glycine dipeptide lipids.

Systemic TLR2 tolerance enhances central nervous system remyelination.

BACKGROUND: Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by both inflammatory demyelination and impaired remyelination. Studies indicate that Toll-like receptor 2 (TLR2) signaling contributes to both the inflammatory component and the defective remyelination in MS. While most MS therapeutics target adaptive immunity, we recently reported that reducing TLR2 signaling in innate immune cells by inducing TLR2 tolerance attenuates adoptively transferred experimental autoimmune encephalomyelitis. Given that previous reports suggest TLR2 signaling also inhibits myelin repair, the objective of this study was to assess how reducing TLR2 signaling through TLR2 tolerance induction affects CNS myelin repair. METHODS: Chow containing 0.2% cuprizone was fed to male and female wild-type (WT) C57BL/6 mice or TLR2-deficient (TLR2-/-) mice for 5 weeks to induce demyelination. During a 2-week remyelination period following discontinuation of cuprizone, WT mice received either low dose TLR2 ligands to induce systemic TLR2 tolerance or vehicle control (VC). Remyelination was evaluated via electron microscopy and immunohistochemical analysis of microglia and oligodendrocytes in the corpus callosum. Statistical tests included 2-way ANOVA and Mann-Whitney U analyses. RESULTS: Inducing TLR2 tolerance in WT mice during remyelination significantly enhanced myelin recovery, restoring unmyelinated axon frequency and myelin thickness to baseline levels compared to VC-treated mice. Mechanistically, enhanced remyelination in TLR2 tolerized mice was associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. This result was confirmed in vitro by inducing TLR2 tolerance in WT microglia cultures. TLR2-/- mice, without TLR2 tolerance induction, also significantly enhanced myelin recovery compared to WT mice, adding confirmation that reduced TLR2 signaling is associated with enhanced remyelination. DISCUSSION: Our results suggest that reducing TLR2 signaling in vivo by inducing TLR2 tolerance significantly enhances myelin repair. Furthermore, the enhanced remyelination resulting from TLR2 tolerance induction is associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. While deletion of TLR2 would be an impractical approach in vivo, reducing innate immune signaling through TLR2 tolerance induction may represent a novel, two-pronged approach for treating both inflammatory and myelin repair components of MS.

ATP increases [Ca2+ ]i and activates a Ca2+ -dependent Cl- current in rat ventricular fibroblasts.

NEW FINDINGS: What is the central question of this study? Although electrophysiological and biophysical characteristics of heart fibroblasts have been studied in detail, their responses to prominent paracrine agents in the myocardium have not been addressed adequately. Our experiments characterize changes in cellular electrophysiology and intracellular calcium in response to ATP. What is the main finding and its importance? In rat ventricular fibroblasts maintained in cell culture, we find that ATP activates a specific subset of Ca2+ -activated Cl- channels as a consequence of binding to P2Y purinoceptors and then activating phospholipase C. This response is not dependent on [Ca2+ ]o but requires an increase in [Ca2+ ]i and is modulated by the type of nucleotide that is the purinergic agonist. ABSTRACT: Effects of ATP on enzymatically isolated rat ventricular fibroblasts maintained in short-term (36-72 h) cell culture were examined. Immunocytochemical staining of these cells revealed that a fibroblast, as opposed to a myofibroblast, phenotype was predominant. ATP, ADP or uridine 5'-triphosphate (UTP) all produced large increases in [Ca2+ ]i . Voltage-clamp studies (amphotericin-perforated patch) showed that ATP (1-100 µm) activated an outwardly rectifying current, with a reversal potential very close to the Nernst potential for Cl- . In contrast, ADP was much less effective, and UTP produced no detectable current. The non-selective Cl- channel blockers niflumic acid, DIDS and NPPB (each at 100 µm), blocked the responses to 100 µm ATP. An agonist for P2Y purinoceptors, 2-MTATP, activated a very similar outwardly rectifying C1- current. The P2Y receptor antagonists, suramin and PPADS (100 µm each), significantly inhibited the Cl- current produced by 100 µm ATP. ATP was able to activate this Cl- current when [Ca2+ ]o was removed, but not when [Ca2+ ]i was buffered with BAPTA-AM. In the presence of the phospholipase C inhibitor U73122, this Cl- current could not be activated. PCR analysis revealed strong signals for a number of P2Y purinoceptors and for the Ca2+ -activated Cl- channel, TMEM16F (also denoted ANO6). In summary, these results demonstrate that activation of P2Y receptors by ATP causes a phospholipase C-dependent increase in [Ca2+ ]i , followed by activation of a Ca2+ -dependent Cl- current in rat ventricular fibroblasts.

TLR Tolerance as a Treatment for Central Nervous System Autoimmunity.

The role of TLR signaling in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is unclear. This role is especially controversial in models of adoptive transfer EAE in which no adjuvant and no TLR ligands are administered. We recently reported that a microbiome-derived TLR2 ligand, Lipid 654 (L654), is present in healthy human serum but significantly decreased in the serum of MS patients. This suggested that microbiome products that gain access to the systemic circulation, rather than being proinflammatory, may normally play an immune-regulatory role by maintaining a state of relative TLR tolerance. Therefore, a loss of microbiome-mediated TLR tolerance, as suggested by lower serum levels of L654, may play a role in the pathogenesis of MS. As proof of concept we asked whether administering low-level TLR2 ligands in adoptive transfer EAE induces TLR2 tolerance and attenuates disease. We administered low-level Pam2CSK4 or L654 to mice receiving encephalitogenic cells and in doing so induced both TLR2 tolerance and attenuation of EAE. Disease attenuation was accompanied in the CNS by a decrease in macrophage activation, a decrease in a specific proinflammatory macrophage population, and a decrease in Th17 cells. In addition, disease attenuation was associated with an increase in splenic type 1 regulatory T cells. Kinetic tolerance induction studies revealed a critical period for TLR2 involvement in adoptive transfer EAE. Overall, these results suggest that inducing TLR tolerance may offer a new approach to treating CNS autoimmune diseases such as MS.

Deposition and hydrolysis of serine dipeptide lipids of Bacteroidetes bacteria in human arteries: relationship to atherosclerosis.

Multiple reaction monitoring-MS analysis of lipid extracts from human carotid endarterectomy and carotid artery samples from young individuals consistently demonstrated the presence of bacterial serine dipeptide lipid classes, including Lipid 654, an agonist for human and mouse Toll-like receptor (TLR)2, and Lipid 430, the deacylated product of Lipid 654. The relative levels of Lipid 654 and Lipid 430 were also determined in common oral and intestinal bacteria from the phylum Bacteroidetes and human serum and brain samples from healthy adults. The median Lipid 430/Lipid 654 ratio observed in carotid endarterectomy samples was significantly higher than the median ratio in lipid extracts of common oral and intestinal Bacteroidetes bacteria, and serum and brain samples from healthy subjects. More importantly, the median Lipid 430/Lipid 654 ratio was significantly elevated in carotid endarterectomies when compared with control artery samples. Our results indicate that deacylation of Lipid 654 to Lipid 430 likely occurs in diseased artery walls due to phospholipase A2 enzyme activity. These results suggest that commensal Bacteriodetes bacteria of the gut and the oral cavity may contribute to the pathogenesis of TLR2-dependent atherosclerosis through serine dipeptide lipid deposition and metabolism in artery walls.

Evaluation of Helping Babies Breathe Quality Improvement Cycle (HBB-QIC) on retention of neonatal resuscitation skills six months after training in Nepal.

BACKGROUND: Each year 700,000 infants die due to intrapartum-related complications. Implementation of Helping Babies Breathe (HBB)-a simplified neonatal resuscitation protocol in low-resource clinical settings has shown to reduce intrapartum stillbirths and first-day neonatal mortality. However, there is a lack of evidence on the effect of different HBB implementation strategies to improve and sustain the clinical competency of health workers on bag-and-mask ventilation. This study was conducted to evaluate the impact of multi-faceted implementation strategy for HBB, as a quality improvement cycle (HBB-QIC), on the retention of neonatal resuscitation skills in a tertiary hospital of Nepal. METHODS: A time-series design was applied. The multi-faceted intervention for HBB-QIC included training, daily bag-and-mask skill checks, preparation for resuscitation before every birth, self-evaluation and peer review on neonatal resuscitation skills, and weekly review meetings. Knowledge and skills were assessed through questionnaires, skill checklists, and Objective Structured Clinical Examinations (OSCE) before implementation of the HBB-QIC, immediately after HBB training, and again at 6 months. Means were compared using paired t-tests, and associations between skill retention and HBB-QIC components were analyzed using logistic regression analysis. RESULTS: One hundred thirty seven health workers were enrolled in the study. Knowledge scores were higher immediately following the HBB training, 16.4 ± 1.4 compared to 12.8 ± 1.6 before (out of 17), and the knowledge was retained 6 months after the training (16.5 ± 1.1). Bag-and-mask skills improved immediately after the training and were retained 6 months after the training. The retention of bag-and-mask skills was associated with daily bag-and-mask skill checks, preparation for resuscitation before every birth, use of a self-evaluation checklist, and attendance at weekly review meetings. The implementation strategies with the highest association to skill retention were daily bag-and-mask skill checks (RR-5.1, 95% CI 1.9-13.5) and use of self-evaluation checklists after every delivery (RR-3.8, 95% CI 1.4-9.7). CONCLUSIONS: Health workers who practiced bag-and-mask skills, prepared for resuscitation before every birth, used self-evaluation checklists, and attended weekly review meetings were more likely to retain their neonatal resuscitation skills. Further studies are required to evaluate HBB-QIC in primary care settings, where the number of deliveries is gradually increasing. TRIAL REGISTRATION: ISRCTN97846009 . Date of Registration- 15 August 2012.

Convergent synthesis of a deuterium-labeled serine dipeptide lipid for analysis of biological samples.

Bacterial serine dipeptide lipids are known to promote inflammatory processes and are detected in human tissues associated with periodontal disease or atherosclerosis. Accurate quantification of bacterial serine lipid, specifically lipid 654 [((S)-15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, (3S)-l-serine] isolated from Porphyromonas gingivalis, in biological samples requires the preparation of a stable isotope internal standard for sample supplementation and subsequent mass spectrometric analysis. This report describes the convergent synthesis of a deuterium-substituted serine dipeptide lipid, which is an isotopically labeled homologue that represents a dominant form of serine dipeptide lipid recovered in bacteria.

Inadequate fetal heart rate monitoring and poor use of partogram associated with intrapartum stillbirth: a case-referent study in Nepal.

BACKGROUND: Newborns are at the greatest risk for dying during the intrapartum period, including labor and delivery, and the first day of life. Fetal heart rate monitoring (FHRM) and partogram use to track labor progress are evidence-based techniques that can help to identify maternal and fetal risk factors so that these can be addressed early. The objective of this study was to assess health worker adherence to protocols for FHRM and partogram use during the intrapartum period, and to assess the association between adherence and intrapartum stillbirth in a tertiary hospital of Nepal. METHODS: A case-referent study was conducted over a 15-month period. Cases included all intrapartum stillbirths, while 20 % of women with live births were randomly selected on admission to make up the referent population. The frequency of FHRM and the use of partogram were measured and their association to intrapartum stillbirth was assessed using logistic regression analysis. RESULTS: During the study period, 4,476 women with live births were enrolled as referents and 136 with intrapartum stillbirths as cases. FHRM every 30 min was only completed in one-fourth of the deliveries, and labor progress was monitored using a partogram in just over half. With decreasing frequency of FHRM, there was an increased risk of intrapartum stillbirth; FHRM at intervals of more than 30 min resulted in a four-fold risk increase for intrapartum stillbirth (aOR 4.17, 95 % CI 2.0-8.7), and the likelihood of intrapartum stillbirth increased seven times if FHRM was performed less than every hour or not at all (aOR 7.38, 95 % CI 3.5-15.4). Additionally, there was a three-fold increased risk of intrapartum stillbirth if the partogram was not used (aOR 3.31, 95 % CI 2.0-5.4). CONCLUSION: The adherence to FHRM and partogram use was inadequate for monitoring intrapartum progress in a tertiary hospital of Nepal. There was an increased risk of intrapartum stillbirth when fetal heart rate was inadequately monitored and when the progress of labor was not monitored using a partogram. Further exploration is required in order to determine and understand the barriers to adherence; and further, to develop tools, techniques and interventions to prevent intrapartum stillbirth. CLINICAL TRIAL REGISTRATION: ISRCTN97846009 .

Incidence of intrapartum stillbirth and associated risk factors in tertiary care setting of Nepal: a case-control study.

BACKGROUND: Each year, 1.2 million intrapartum stillbirths occur globally. In Nepal, about 50 % of the total number of stillbirths occur during the intrapartum period. An understanding of the risk factors associated with intrapartum stillbirth will facilitate the development of preventative strategies to reduce the associated burden of death. This study was conducted in a tertiary-care setting with the aim to identify risk factors associated with intrapartum stillbirth. METHODS: A case-control study was completed from July 2012 to September 2013. All women who had an intrapartum stillbirth during the study period were included as cases, and 20 % of women with live births were randomly selected upon admission to create the referent population. Relevant information was retrieved from clinical records for case and referent women. In addition, interviews were completed with each woman to determine their demographic and obstetric history. RESULTS: During the study period, 4,476 women were enrolled as referents and 136 women had intrapartum stillbirths. The following factors were found to be associated with an increased risk for intrapartum stillbirth: poor familial wealth quintile (Adj OR 1.8, 95 % CI-1.1-3.4); less maternal education (Adj OR, 3.2 95 % CI-1.8-5.5); lack of antenatal care (Adj OR, 4.8 95 % CI 3.2-7.2); antepartum hemorrhage (Adj OR 2.1, 95 % CI 1.1-4.2); multiple births (Adj. OR-3.0, 95 % CI- 1.9-5.4); obstetric complication during labor (Adj. OR 4.5, 95 % CI-2.9-6.9); lack of fetal heart rate monitoring per protocol (Adj. OR-1.9, 95 % CI 1.5-2.4); lack of partogram use (Adj. OR-2.1, 95 % CI 1.1-4.1); small-for-gestational age (Adj. OR-1.8, 95 % CI-1.2-1.7); preterm birth (Adj. OR-5.4, 95 % CI 3.5-8.2); and being born preterm with a small-for-gestational age (Adj. OR-9.0, 95 % CI 7.3-15.5). CONCLUSION: Being born preterm with a small-for-gestational age was associated with the highest risk for intrapartum stillbirth. Inadequate fetal heart rate monitoring and partogram use are preventable risk factors associated with intrapartum stillbirth; by increasing adherence to these interventions the risk of intrapartum stillbirth can be reduced. The association of the lack of appropriate antenatal care with intrapartum stillbirth indicates that quality antenatal care may improve fetal health and outcomes. TRIAL REGISTRATION: ISRCTN97846009.

Cbl-b-deficient mice express alterations in trafficking-related molecules but retain sensitivity to the multiple sclerosis therapeutic agent, FTY720.

The variable response to therapy in multiple sclerosis (MS) suggests a need for personalized approaches based on individual genetic differences. GWAS have linked CBLB gene polymorphisms with MS and recent evidence demonstrated that these polymorphisms can be associated with abnormalities in T cell function and response to interferon-ß therapy. Cbl-b is an E3 ubiquitin ligase that regulates T cell activation and Cbl-b-deficient (Cbl-b(-/-)) mice show T cell abnormalities described in MS patients. We now show that Cbl-b(-/-) T cells demonstrate significant lymph node trafficking abnormalities. We thus asked whether the MS-approved drug, FTY720, postulated to trap T cells in lymphoid tissues, is less effective in the context of Cbl-b dysfunction. We now report that FTY720 significantly inhibits EAE in Cbl-b(-/-) mice. Our results newly document a role for Cbl-b in T cell trafficking but suggest nevertheless that MS patients with Cbl-b abnormalities may still be excellent candidates for FTY720 treatment.

Phospholamban knockout breaks arrhythmogenic Ca²âº waves and suppresses catecholaminergic polymorphic ventricular tachycardia in mice.

RATIONALE: Phospholamban (PLN) is an inhibitor of cardiac sarco(endo)plasmic reticulum Ca²âº ATPase. PLN knockout (PLN-KO) enhances sarcoplasmic reticulum Ca²âº load and Ca²âº leak. Conversely, PLN-KO accelerates Ca²âº sequestration and aborts arrhythmogenic spontaneous Ca²âº waves (SCWs). An important question is whether these seemingly paradoxical effects of PLN-KO exacerbate or protect against Ca²âº-triggered arrhythmias. OBJECTIVE: We investigate the impact of PLN-KO on SCWs, triggered activities, and stress-induced ventricular tachyarrhythmias (VTs) in a mouse model of cardiac ryanodine-receptor (RyR2)-linked catecholaminergic polymorphic VT. METHODS AND RESULTS: We generated a PLN-deficient, RyR2-mutant mouse model (PLN-/-/RyR2-R4496C+/-) by crossbreeding PLN-KO mice with catecholaminergic polymorphic VT-associated RyR2-R4496C mutant mice. Ca²âº imaging and patch-clamp recording revealed cell-wide propagating SCWs and triggered activities in RyR2-R4496C+/- ventricular myocytes during sarcoplasmic reticulum Ca²âº overload. PLN-KO fragmented these cell-wide SCWs into mini-waves and Ca²âº sparks and suppressed the triggered activities evoked by sarcoplasmic reticulum Ca²âº overload. Importantly, these effects of PLN-KO were reverted by partially inhibiting sarco(endo)plasmic reticulum Ca²âº ATPase with 2,5-di-tert-butylhydroquinone. However, Bay K, caffeine, or Li⁺ failed to convert mini-waves to cell-wide SCWs in PLN-/-/RyR2-R4496C+/- ventricular myocytes. Furthermore, ECG analysis showed that PLN-KO mice are not susceptible to stress-induced VTs. On the contrary, PLN-KO protected RyR2-R4496C mutant mice from stress-induced VTs. CONCLUSIONS: Our results demonstrate that despite severe sarcoplasmic reticulum Ca²âº leak, PLN-KO suppresses triggered activities and stress-induced VTs in a mouse model of catecholaminergic polymorphic VT. These data suggest that breaking up cell-wide propagating SCWs by enhancing Ca²âº sequestration represents an effective approach for suppressing Ca²âº-triggered arrhythmias.

Regulatory T-cells and cAMP suppress effector T-cells independently of PKA-CREM/ICER: a potential role for Epac.

cAMP signalling is both a major pathway as well as a key therapeutic target for inducing immune tolerance and is involved in Treg cell (regulatory T-cell) function. To achieve potent immunoregulation, cAMP can act through several downstream effectors. One proposed mechanism is that cAMP-mediated suppression, including immunosuppression by Treg cells, results from activation of PKA (protein kinase A) leading to the induction of the transcription factor ICER (inducible cAMP early repressor). In the present study, we examined CD4(+)CD25(-) Teff cell (effector T-cell) and CD4(+)CD25(+) Treg cell immune responses in Crem (cAMP-response-element modulator) gene-deficient mice which lack ICER (Crem(-/-)/ICER-deficient mice). ICER deficiency did not significantly alter the frequency or number of Treg cells and Teff cells. Treg cells or a pharmacological increase in cAMP suppressed Teff cells from Crem(+/+) and Crem(-/-)/ICER-deficient mice to an equivalent degree, demonstrating that ICER is dispensable in these functions. Additionally, activating the cAMP effector Epac (exchange protein directly activated by cAMP) suppressed Teff cells. Treg cells expressed low levels of all cyclic nucleotide Pde (phosphodiesterase) genes tested, but high levels of Epac. These data identify ICER as a redundant mediator of Treg cells and cAMP action on Teff cells and suggest that Epac may function as an alternative effector to promote cAMP-dependent Teff cell suppression.

The role of chromogranin B in an animal model of multiple sclerosis.

Chromogranin B (CGB) is a high capacity, low affinity calcium binding protein in the endoplasmic reticulum (ER) that binds to the inositol 1,4,5 trisphosphate receptor (InsP3R) and amplifies calcium release from ER stores. Recently, it was discovered that levels of CGB-derived peptides are decreased in the cerebrospinal fluid of multiple sclerosis (MS) patients. One of the mechanisms by which neurodegeneration in MS is thought to occur is through increased levels of intra-axonal calcium. The combination of excess intracellular calcium and dysregulated levels of CGB in MS led us to hypothesize that CGB may be involved in MS pathophysiology. Here, we show in a mouse model of MS that CGB levels are elevated in neurons prior to onset of symptoms. Once symptoms develop, CGB protein levels increase with disease severity. Additionally, we show that elevated levels of CGB may have a role in the pathophysiology of MS and suggest that the initial elevation of CGB, prior to symptom onset, is due to inflammatory processes. Upon development of symptoms, CGB accumulation in neurons results from decreased ubiquitination and decreased secretion. Furthermore, we show that calpain activity is increased and levels of InsP3R are decreased. From these results, we suggest that the elevated levels of CGB and altered InsP3R levels may contribute to the axonal/neuronal damage and dysregulated calcium homeostasis observed in MS. Additionally, we propose that CGB can be a biomarker that predicts the onset and severity of disease in patients with MS.

Microbiome-derived bacterial lipids regulate gene expression of proinflammatory pathway inhibitors in systemic monocytes.

How the microbiome regulates responses of systemic innate immune cells is unclear. In the present study, our purpose was to document a novel mechanism by which the microbiome mediates crosstalk with the systemic innate immune system. We have identified a family of microbiome Bacteroidota-derived lipopeptides-the serine-glycine (S/G) lipids, which are TLR2 ligands, access the systemic circulation, and regulate proinflammatory responses of splenic monocytes. To document the role of these lipids in regulating systemic immunity, we used oral gavage with an antibiotic to decrease the production of these lipids and administered exogenously purified lipids to increase the systemic level of these lipids. We found that decreasing systemic S/G lipids by decreasing microbiome Bacteroidota significantly enhanced splenic monocyte proinflammatory responses. Replenishing systemic levels of S/G lipids via exogenous administration returned splenic monocyte responses to control levels. Transcriptomic analysis demonstrated that S/G lipids regulate monocyte proinflammatory responses at the level of gene expression of a small set of upstream inhibitors of TLR and NF-κB pathways that include Trem2 and Irf4. Consistent with enhancement in proinflammatory cytokine responses, decreasing S/G lipids lowered gene expression of specific pathway inhibitors. Replenishing S/G lipids normalized gene expression of these inhibitors. In conclusion, our results suggest that microbiome-derived S/G lipids normally establish a level of buffered signaling activation necessary for well-regulated innate immune responses in systemic monocytes. By regulating gene expression of inflammatory pathway inhibitors such as Trem2, S/G lipids merit broader investigation into the potential dysfunction of other innate immune cells, such as microglia, in diseases such as Alzheimer's disease.

Serine lipids of Porphyromonas gingivalis are human and mouse Toll-like receptor 2 ligands.

The total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. In vitro, both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2(-/-)) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2(-/-) mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced by P. gingivalis that likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate.

Natural but not inducible regulatory T cells require TNF-alpha signaling for in vivo function.

TNF-α has a multifunctional role in autoimmune diseases as reflected in the variable responses of different human diseases to anti-TNF-α therapy. Recent studies have suggested that TNF-α modulates autoimmunity partially via effects on regulatory T cells (Tregs) and that these effects are mediated through the type II TNFR (TNFR2). We have investigated the requirement for TNFR2-expression on murine natural Tregs (nTregs) and induced Tregs (iTregs) in mediating suppression of colitis. Surprisingly, we find that TNFR2-expression is required for both spleen- and thymus-derived nTreg-mediated suppression, but is not required for iTreg-mediated suppression. Abnormal TNFR2(-/-) nTreg function was not associated with an in vivo decrease in accumulation, stability, or expression of markers known to be relevant in Treg function. Because iTregs are generated in the presence of TGF-ß, we investigated whether activation in the presence of TGF-ß could overcome the functional defect in TNFR2(-/-) nTregs. Although preactivation alone did not restore suppressive function of nTregs, preactivation in the presence of TGF-ß did. These results identify potentially critical differences in activation requirements for nTregs versus iTregs. Furthermore, our findings are consistent with reports suggesting that nTregs are activated in sites of inflammation while iTregs are activated in lymph nodes. Finally, by demonstrating that nTregs require TNF-α for optimal function whereas iTregs do not, our results suggest that the enigma of variable responses of different human diseases to anti-TNF-α therapy may relate to whether nTregs or iTregs have the predominant regulatory role in a given disease.