Experimental Endotoxemia Research Articles

Changes in microcirculation variables in an acute endotoxaemic equine model.

Microcirculation is the essential link between macrocirculation and cellular metabolism. To test our hypotheses that microcirculation variables will show a heterogeneous flow pattern during experimental endotoxaemia, and that fluid therapy and noradrenaline (NA) infusion will normalise altered microcirculation variables. In vivo experiments. Six healthy adult horses were anaesthetised with dexmedetomidine, ketamine, and diazepam and were mechanically ventilated under isoflurane anaesthesia. Endotoxaemia was induced with 30 ng kg-1 Escherichia coli lipopolysaccharide intravenously. One hundred and twenty minutes later fluid bolus and noradrenaline (NA) infusion were administered to produce normotension. Pulse rate (PR) and mean arterial blood pressure (MAP) were measured and microcirculation variables were obtained by side-stream darkfield technique (de Backer density (DBD), perfused de Backer density (PDBD), proportion of perfused vessels, microvascular flow index (MFI), heterogeneity index (HI)), laser Doppler flowmetry (blood flow) and white light spectrometry (tissue oxygen saturation (tSO2)) in sublingual, jejunal and genital area. Measurements were obtained at baseline, after endotoxin, at 60 and 120 min and during the normotensive phase. Data were analysed by mixed model variance analysis and Tukey-Kramer. The PPV decreased significantly over time by 30% (p < 0.001) at the jejunum. MFI decreased from baseline to ET60 and from baseline to ET120 in sublingual and genital mucosa (2.9 vs. 1.4, p < 0.001 and 2.8 vs. 1.9, p < 0.01), respectively. The sublingual HI increased from baseline to ET60, ET120 and NA (0.1 vs. 0.9, p = 0.02; vs. 0.6, p = 0.01; vs. 0.3, p = 0.01), respectively. The genital HI increased from baseline to ET120 (0.2 vs. 1.1, p ≤ 0.01) and NA (0.16 vs. 0.53, p < 0.05, respectively). Moderate agreement between observers for MFI assessment was present (kappa = 0.4). The PR significantly increased, and MAP significantly decreased from baseline over time. The obtained data could be influenced by secretions, pressure artefacts, the experience of the examiner and the sampling location. Blood flow was not quantified and there was no control group. Overall, short-term experimental endotoxaemia did negatively alter MFI and HI; however, it did not alter tSO2, blood flow, DBD, PDBD or proportion of perfused vessels. Intravenous fluid therapy and NA did not restore MFI and HI to baseline values.

Hemodynamic Response to Lipopolysaccharide Infusion and Effect of Meloxicam Administration on Cardiac Function in Donkeys.

Systemic inflammatory response syndrome (SIRS) in donkeys is observed to be secondary to colic, diarrhea or pleuropneumonia, among other disorders. Horses with SIRS develop secondary disturbances such as hyperlipemia, laminitis, disseminated intravascular coagulopathy, and hemodynamic and cardiac derangements, which impair their prognosis and increase the mortality rate. In donkeys, no information is available on the effect of experimentally induced endotoxemia in the cardiovascular system. Acute experimental endotoxemia was induced by lipopolysaccharide (LPS) infusion in six healthy adult non-pregnant jennies. Physical signs, arterial (systolic, diastolic and mean) and central venous pressure were monitored during 360 min. Cardiac troponin I (cTnI) concentrations were measured in blood samples, and echocardiography was performed. LPS infusion caused an increase in cTnI, hypotension and diminution of central venous pressure, cardiac dysfunction, with a decrease in stroke volume (SV), cardiac output (CO) and cardiac index, and impairment of ultrasonographic ventricular function parameters. Intravenous meloxicam administration prevented the cTnI increase, hypotension, diminution of SV and CO, and changes in ultrasonographic parameters related to ventricular dysfunction. Thus, meloxicam could be proposed as an effective therapeutical option to control the hemodynamic and cardiac derangements observed in donkeys with SIRS.

Investigation of the effects of melatonin on lung tissue through the NLRP3/TLR2/NEK7 pathway in an experimental endotoxemia model.

Sepsis, a severe clinical syndrome, arises from pro-inflammatory and apoptotic processes. Its rapid progression from sepsis to severe stages necessitates timely intervention. The lipopolysaccharide (LPS) agent triggers pro-inflammatory mediator release through Toll-like receptors, particularly TLR-2, a vital biomarker in sepsis with multiple organ failure. In LPS-induced septic shock, the NEK7-mediated NLRP3 inflammasome pathway, linked to acute lung injury, is suppressed. This pathway is implicated in sepsis-induced platelet activation and septic shock development. Antioxidants like melatonin (MEL) may positively impact reducing septic shock. In microbial-induced sepsis, melatonin can regulate pro-inflammatory mediator transcriptional activation, potentially controlling the pro-inflammatory state. In the project, the histopathological impact of melatonin in lung tissue during endotoxic shock induced by the LPS agent in Sprague-Dawley rats, and its immunoreactivity to NLRP3/NEK7/TLR-2 molecules, were assessed. Lung volumes were evaluated using micro-computed tomography (Micro-CT). While bleeding, cell infiltration, and thickening of the alveolar wall were observed in the lungs of the LPS group, a reduction in these symptoms was noted in the MEL+LPS group. Expressions of NEK7, TLR2, and NLRP3 increased in both the LPS and MEL+LPS groups compared to the control group. It was determined that in the MEL+LPS group, levels of NEK7, TLR2, and Malondialdehyde (MDA) decreased compared to the LPS group. Additionally, a decrease in the total volume of lung tissue was observed in the LPS group. In this context, our study reported the therapeutic effect of melatonin on sepsis-related acute lung injury. Our study suggests that melatonin administration in the experimental endotoxemia model melatonin may help reduce lung damage by inhibiting NEK7 and TLR2 expressions.

1-Piperidine Propionic Acid Protects from Septic Shock Through Protease Receptor 2 Inhibition.

Sepsis is a complex disorder caused by a dysregulated host response to infection, with high levels of morbidity and mortality. Treatment aimed to modulate immune response and maintain vascular function is still one of the major clinical challenges. This study was designed to test the effect of the small molecule 1-Piperidine Propionic Acid (1-PPA) as molecular targeted agent to block protease-activated receptor 2 (PAR2), one of the major modulators of inflammatory response in LPS-induced experimental endotoxemia. In the THP-1 cell line, LPS-induced cytokine expression was inhibited by 1-PPA in a dose-dependent manner. In LPS-injected mice, treatment with 1-PPA was effective in reducing mortality and sepsis-related symptoms and improved cardiac function parameters. After 6 h from LPS injection, a significant decrease in IL-6, IL-1β, and IL-10 was observed in the lung tissue of 1-PPA-treated mice, compared to controls. In these mice, a significant decrease in vasoactive molecules, especially kininogen-1, was also observed, mainly in the liver. Histopathological analysis confirmed typical features of sepsis in different organs and these findings were markedly reduced in mice treated with 1-PPA. These data demonstrate the effectiveness of 1-PPA in protecting the whole organism from sepsis-induced damage.

Гістоструктурні зміни в імунокомпетентних органах, печінці та легенях за умов експериментальної ендотоксемії, індукованої ліпополісахаридом

The purpose of the work was to study morphological changes in immunocompetent organs, liver and lungs during experimental endotoxemia induced by lipopolysaccharide (LPS). The histological method with hematoxylin-eosin staining and subsequent microscopic analysis was used in the work. The histostructural damage to the immune system organs (thymus, spleen, lymph nodes) occurs in experimental endotoxemia caused by LPS. There is a circulatory disorder with morphological changes in all layers of the vascular walls. Necrobiotic and necrotic damage to organ cells was also revealed. The liver of experimental animals underwent especially pronounced changes in histostructure. Significant microcirculation disorders with dilation and congestion of vessels of various sizes and significant damage to endothelial cells were identified. Necrotic changes in hepatocytes were accompanied by a significant increase in stellate reticuloendotheliocytes and increased lymphocytic infiltration, which is the basis for liver dysfunction and the development of an inflammatory immune reaction. In conclusion, the data obtained on the morphological manifestations of endotoxemic damage can serve as the basis for the development of effective therapeutic approaches in the treatment of LPS-induced internal organ damage.

Hemostatic conditions following autologous transfusion of fresh vs stored platelets in experimental endotoxemia: an open-label randomized controlled trial with healthy volunteers

BackgroundPlatelet increment is reportedly lower for maximum stored platelet concentrates (PCs) and during pyrexia, and in vitro function differs between fresh and stored PCs. However, little is known about the function of fresh and stored platelets during inflammation. ObjectivesThe aim was to study differences in hemostatic function after transfusion of fresh or stored PCs in a human model of experimental endotoxemia. MethodsThirty-six healthy male subjects received either 2 ng/kg lipopolysaccharide (LPS) or a control (physiological saline 0.9%) and were randomly assigned to subsequently receive an autologous transfusion of either fresh (2-days-old) or stored (7-days-old) platelets, or saline control. Extracellular vesicles (EVs) were determined using flow cytometry, thrombin–antithrombin complex (TATc) was assessed using enzyme-linked immunosorbent assay, and hemostatic function was assessed using rotational thromboelastometry (ROTEM). ResultsLPS infusion caused a marked increase in TATc, EVs and fibrinolysis. Thromboelastometry data revealed that following infusion of LPS, subjects exhibited in general a hypocoagulable state compared with those not receiving LPS. Platelet transfusions led to a reduced clotting time and an augmentation in clot strength, indicated by maximum clot firmness, solely among subjects undergoing endotoxemia. There were no significant differences in TATc or amount of EVs release after transfusion of fresh or stored platelets. ConclusionA significant increase in TATc and EVs as well as a difference in hemostatic function after endotoxemia were observed. During endotoxemia, platelet transfusion resulted in enhanced coagulation and hemostatic function; however, no substantial differences were observed between transfusion of fresh or stored PCs.

The possible cardioprotective effect of ghrelin during experimental endotoxemia in mice.

This study aimed to evaluate the cardioprotective effects of ghrelin in septic mice, focusing on its anti-inflammatory and antioxidant properties. Thirty-five male Swiss mice (8-12 weeks old, 23-33g) were randomly assigned to five groups (n = 7 each): (1) Normal, fed usual diets, (2) Sham, subjected to anesthesia and laparotomy, (3) Sepsis, subjected to cecal ligation and puncture, (4) Vehicle, given an equivalent volume of intraperitoneal saline injections immediately after cecal ligation and puncture, and (5) Ghrelin-treated, administered 80 µg/kg ghrelin intraperitoneal injections immediately following cecal ligation and puncture. Serum levels of tumor necrosis factor-alpha (TNF-α), macrophage migration inhibitory factor (MIF), toll-like receptor 4 (TLR4), and 8-epi-prostaglandin F2 alpha (8-epi-PGF2α) were measured. The extent of cardiac damage was also evaluated histologically. The mean serum levels of TNF-α, MIF, TLR4, and 8-epi-PGF2α levels were significantly higher in the sepsis and vehicle groups than in the normal and sham groups. The levels were significantly lower in the ghrelin-treated group than in the vehicle and sepsis groups. Histological analysis revealed normal myocardial architecture in the normal and sham groups, whereas the sepsis and vehicle groups had severe myocardial injury. The ghrelin-treated group displayed histological features similar to the sham group, indicating reduced myocardial damage. Ghrelin ameliorated sepsis-induced cardiotoxicity in mice by exhibiting strong anti-inflammatory and antioxidant effects. These findings suggest that ghrelin may be a promising therapeutic candidate for the prevention of sepsis-induced cardiotoxicity.

Perception of unfamiliar caregivers during sickness – Using the new Caregiver Perception Task (CgPT) during experimental endotoxemia

Social withdrawal is a well-established part of sickness behavior, but in some contexts sick animals might gain from keeping close instead of keeping away. For instance, sick individuals are more willing to be near known individuals who can provide care and safety (close others) compared to when healthy. Yet, interactions with some strangers might also be beneficial (i.e., healthcare professionals), but it is not known how sickness interplay with social behavior towards such individuals. Here, we assessed if sickness affects perception of caregivers, and developed a new task, the Caregiver Perception Task (CgPT). Twenty-six participants performed the CgPT, once after an injection of lipopolysaccharide (LPS, 0.8 ng/kg body weight, n = 24), and once after an injection of saline (n = 25), one hour and forty-five minutes post-injection. During the task, participants watched short video clips of three types of caregivers: a healthcare professional taking care of a sick individual, a healthcare professional not taking care of a sick individual, and a non-healthcare professional taking care of their sick adult child or partner. After each video clip, the likability, trustworthiness, professionalism, and willingness to interact with and receive care from the caregiver were rated on visual analogue scales. Results showed that participants injected with saline rated healthcare professionals who did not take care of a sick individual less positively on all aspects compared to healthcare professionals who took care of a sick individual. Moreover, compared to saline, LPS increased the participants’ willingness to receive care from healthcare professionals and non-healthcare professionals providing care, but not from healthcare professionals not providing care. Thus, our results indicate that sick individuals may approach unknown individuals with potential to provide care and support.

Germanium citrate improves ovarian granulosa cells viability and antioxidant defense system in aging female mice during endotoxemia

The study aimed to investigate the vitality of ovarian granulosa cells (GCs), metabolic activity of neutrophils, as well as the antioxidant system state in aging female mice subjected to experimental endotoxemia, as well as the influence of germanium (Ge) citrate on the studied parameters under these conditions. Treatment with Gramm-negative bacteria lipopolysaccharide caused pathological changes in mouse ovaries: a decrease in GCs viability by increasing necrosis, an enhancement of metabolic activity of peripheral blood neutrophils, an increase in lipid peroxidaxion products and a violation of the antioxidant defense system (as evidenced by an elevation of the content of reactive products of 2-thiobarbituric acid and a reduction of reduced glutathione in liver homogenate, as well as a decrease in the concentration of ceruloplasmin in blood serum of aging female mice). Pretreatment of mice with Ge citrate was effective to reduce GCs death and improve their viability, decrease the degree of disruption of the redox balance and weaken the activity of cells of non-specific immune protection in aging endotoxemic animals. Our results suggest that Ge citrate may offer promising therapeutic benefits. Its cytoprotective effects and regulatory role in the antioxidant defense system, combined with its potential to reduce the intensity of gram-negative bacterial toxins induced inflammation, imply its usefulness. This multifaceted action may help prevent ovarian cell aging and, consequently, improve reproductive function.

Myeloid-Derived Suppressor-like Cells as a Prognostic Marker in Critically Ill Patients: Insights from Experimental Endotoxemia and Intensive Care Patients.

Patients admitted to the intensive care unit (ICU) often experience endotoxemia, nosocomial infections and sepsis. Polymorphonuclear and monocytic myeloid-derived suppressor cells (PMN-MDSCs and M-MDSCs) can have an important impact on the development of infectious diseases, but little is known about their potential predictive value in critically ill patients. Here, we used unsupervised flow cytometry analyses to quantify MDSC-like cells in healthy subjects challenged with endotoxin and in critically ill patients admitted to intensive care units and at risk of developing infections. Cells phenotypically similar to PMN-MDSCs and M-MDSCs increased after endotoxin challenge. Similar cells were elevated in patients at ICU admission and normalized at ICU discharge. A subpopulation of M-MDSC-like cells expressing intermediate levels of CD15 (CD15int M-MDSCs) was associated with overall mortality (p = 0.02). Interestingly, the high abundance of PMN-MDSCs and CD15int M-MDSCs was a good predictor of mortality (p = 0.0046 and 0.014), with area under the ROC curve for mortality of 0.70 (95% CI = 0.4-1.0) and 0.86 (0.62-1.0), respectively. Overall, our observations support the idea that MDSCs represent biomarkers for sepsis and that flow cytometry monitoring of MDSCs may be used to risk-stratify ICU patients for targeted therapy.

Altered response to Toll-like receptor 4 activation in fibromyalgia: A low-dose, human experimental endotoxemia pilot study

In this pilot study, a human intravenous injection of low-dose endotoxin (lipopolysaccharide, LPS) model was used to test if fibromyalgia is associated with altered immune responses to Toll-like receptor 4 (TLR4) activation. Eight women with moderately-severe fibromyalgia and eight healthy women were administered LPS at 0.1 ng/kg in session one and 0.4 ng/kg in session two. Blood draws were collected hourly to characterize the immune response. The primary analytes of interest, leptin and fractalkine, were assayed via commercial radioimmunoassay and enzyme-linked immunosorbent assay kits, respectively. Exploratory analyses were performed on 20 secreted cytokine assays by multiplex cytokine panels, collected hourly. Exploratory analyses were also performed on testosterone, estrogen, and cortisol levels, collected hourly. Additionally, standard clinical complete blood counts with differential (CBC-D) were collected before LPS administration and at the end of the session. The fibromyalgia group demonstrated enhanced leptin and suppressed fractalkine responses to LPS administration. In the exploratory analyses, the fibromyalgia group showed a lower release of IFN-γ, CXCL10, IL-17A, and IL-12 and higher release of IL-15, TARC, MDC, and eotaxin than the healthy group. The results of this study suggest that fibromyalgia may involve an altered immune response to TLR4 activation.

Interactions between aspirin and ticagrelor during experimental human endotoxaemia

Abstract Introduction Dual antiplatelet therapy (DAPT) with aspirin 75-100mg once daily (OD) and the P2Y12 inhibitor ticagrelor is a standard treatment for acute coronary syndromes. De-escalating DAPT to ticagrelor monotherapy reduces bleeding risk yet appears to have minimal ischaemic penalty in most groups. The reason for this is unclear. P2Y12 inhibition can inhibit inflammatory processes relevant to atherothrombosis but aspirin may hypothetically counter these via platelet PGE2 inhibition. We studied the regimen-dependent effects of aspirin +/- ticagrelor in a human model of endotoxaemia. Endotoxin activates toll-like receptor 4, a key damage receptor in atherogenesis. Methods 72 healthy volunteers were randomised to receive either no aspirin or aspirin 20mg twice daily (BD), 75mg OD or 300mg OD for 10 days +/- a loading dose of ticagrelor on the last day prior to receiving intravenous endotoxin administration (2ng/kg). Blood was collected at 9 timepoints from 1h pre to 6h post endotoxin. Key cytokines and prostanoids in the blood/urine were measured by ELISA, platelet function by light transmittance aggregometry and platelet-leukocyte interaction by flow cytometry. Bleeding time was measured pre and 3h post endotoxin. After &amp;gt;5 weeks, in a 2nd period they received the same dose of aspirin as the 1st but received ticagrelor if they had not already, and vice versa, undergoing a 2nd endotoxin challenge at the end of the period. The primary endpoint was plasma tumour necrosis factor (TNF)-α. Results 72 participants were randomised (median 22.5yrs [range 18-59], 6F:66M). A total of 128 endotoxin challenges were performed. Endotoxaemia was associated with flu-like symptoms, pyrexia, tachycardia and relative hypotension but was safe. In those not receiving aspirin, plasma TNF-α was significantly lower when receiving ticagrelor vs. no ticagrelor, but in those receiving any dose of aspirin the significant effect of ticagrelor was abolished (Fig 1). All doses of aspirin fully suppressed arachidonic acid-induced PA with or without ticagrelor despite the thromboinflammatory stimulus of endotoxaemia (Fig 2A), and significantly augmented the inhibitory effect of ticagrelor on collagen-induced PA (2B). Aspirin 75mg OD and 300mg OD, but not 20mg BD, significantly prolonged bleeding time from baseline (2C). Ticagrelor significantly prolonged bleeding time in combination with any dose of aspirin or no aspirin. Aspirin dose-dependently inhibited PGE2 release. Conclusions All doses of aspirin tested prevented the ability of ticagrelor to significantly inhibit cytokine release during experimental endotoxaemia. Aspirin 20mg BD may offer improved bleeding risk with maintained antiplatelet effect compared to standard regimens. Where ticagrelor monotherapy is an option, dropping aspirin may allow ticagrelor to exert greater beneficial effect on inflammatory pathways relevant to atherothrombosis, potentially reducing ischaemic risk whilst also reducing bleeding risk.Figure 1Figure 2

Cytokine Adsorption Effects of a Novel Hemofiltration Column for the Treatment of Experimental Endotoxemia

Introduction: The TKM-101 is a new hemofiltration column packed with a polymer alloy membrane consisting of polyethersulfone, polyvinylpyrrolidone, and sulfonated poly (arylene ether) copolymers. We examined the ability of the TKM-101 column to remove cytokines and humoral mediators from blood in vitro and the effects of extracorporeal treatment with the TKM-101 column on the mortality rate and inflammatory responses to endotoxic shock in vivo. Methods: In vitro and in vivo laboratory investigations were conducted. In the in vitro experiment, the adsorption abilities of TKM-101, AN69-ST, and control columns for cytokine-related sepsis in blood were compared using human serum samples. In the in vivo experiment, male Sprague-Dawley rats were anesthetized and injected with Escherichia coli endotoxin (15 mg/kg, intravenously). Afterward, the rats were assigned (in a double-blind manner) to one of three groups (n = 17 per group): apheresis with a control column (control group), apheresis with an AN69-ST column (AN69-ST group), or apheresis with a TKM-101 column (TKM-101 group). Outcomes were compared among the groups. Results: In vitro, the concentrations of all evaluated cytokines significantly decreased with the TKM-101 column compared to those with the control column; however, there were no significant differences between the TKM-101 and AN69-ST columns. In vivo, the mortality rates 8 h after endotoxin injection were 65%, 29%, and 29% for the control, AN69-ST, and TKM-101 groups, respectively. Hypotension and elevated plasma cytokine concentrations were less prominent in the TKM-101 and AN69-ST groups compared to those in the control group. Conclusions: TKM-101 effectively removed proteins of varying sizes, from small-sized proteins such as interleukin (IL)-8 to mid-sized protein such as IL-10 in vitro. Moreover, TKM-101 treatment reduced mortality and had inhibitory effects on inflammatory responses in endotoxemic rats. These findings suggest that TKM-101 treatment may be available for use in patients with sepsis and/or endotoxemia.

Barrier-protective properties of pterostilbene against LPS-induced sepsis

Background: Sepsis is a major clinical problem and the leading cause of death in patients in intensive care units worldwide. Objective: The present study aimed to explore whether pterostilbene (PTS) could protect mice against experimental endotoxemia. Methodology: Forty-eight mice were randomly divided into 4 groups and challenged with LPS. Mouse mortality was observed twice daily for 7 days and survival rates were reported. Mice were randomly treated with pterostilbene or vehicle intraperitoneally (i.p.). One hour later, the animals were exposed to LPS (20 mg/kg). Cytokine responses were then assessed in serum isolated from blood collected after LPS administration to the mice. Results: The results showed that pterostilbene significantly reduced mouse body weight loss and attenuated inflammatory responses by inhibiting TNF-α, IL-6 and IL-1β production in mice exposed to LPS. Conclusion: This study highlights the role of pterostilbene in the pathogenesis of LPS-induced sepsis and its potential in the treatment of patients with sepsis.

A novel TWIK2 channel inhibitor binds at the bottom of the selectivity filter and protects against LPS-induced experimental endotoxemia in vivo

TWIK2 channel plays a critical role in NLRP3 inflammasome activation and mice deficient in TWIK2 channel are protected from sepsis and inflammatory lung injury. However, inhibitors of TWIK2 channel are currently in an early stage of development, and the molecular determinants underlying the chemical modulation of TWIK2 channel remain unexplored. In this study, we identified NPBA and the synthesized derivative NPBA-4 potently and selectively inhibited TWIK2 channel by using whole-cell patch clamp techniques. Furthermore, the mutation of the last residues of the selectivity filter in both P1 and P2 (i.e., T106A, T214A) of TWIK2 channel substantially abolished the effect of NPBA on TWIK2 channel. Our data suggest that NPBA blocked TWIK2 channel through binding at the bottom of the selectivity filter, which was also supported by molecular docking prediction. Moreover, we found that NPBA significantly suppressed NLRP3 inflammasome activation in macrophages and alleviated LPS-induced endotoxemia and organ injury in vivo. Notably, the protective effects of NPBA against LPS-induced endotoxemia were abolished in Kcnk6-/- mice. In summary, our study has uncovered a series of novel inhibitors of TWIK2 channel and revealed their distinct molecular determinants interacting TWIK2 channel. These findings provide new insights into the mechanisms of pharmacological action on TWIK2 channel and opportunities for the development of selective TWIK2 channel modulators to treat related inflammatory diseases.

Immunosuppressive effects of circulating bile acids in human endotoxemia and septic shock: patients with liver failure are at risk

BackgroundSepsis-induced immunosuppression is a frequent cause of opportunistic infections and death in critically ill patients. A better understanding of the underlying mechanisms is needed to develop targeted therapies. Circulating bile acids with immunosuppressive effects were recently identified in critically ill patients. These bile acids activate the monocyte G-protein coupled receptor TGR5, thereby inducing profound innate immune dysfunction. Whether these mechanisms contribute to immunosuppression and disease severity in sepsis is unknown. The aim of this study was to determine if immunosuppressive bile acids are present in endotoxemia and septic shock and, if so, which patients are particularly at risk.MethodsTo induce experimental endotoxemia in humans, ten healthy volunteers received 2 ng/kg E. coli lipopolysaccharide (LPS). Circulating bile acids were profiled before and after LPS administration. Furthermore, 48 patients with early (shock onset within < 24 h) and severe septic shock (norepinephrine dose > 0.4 μg/kg/min) and 48 healthy age- and sex-matched controls were analyzed for circulating bile acids. To screen for immunosuppressive effects of circulating bile acids, the capability to induce TGR5 activation was computed for each individual bile acid profile by a recently published formula.ResultsAlthough experimental endotoxemia as well as septic shock led to significant increases in total bile acids compared to controls, this increase was mild in most cases. By contrast, there was a marked and significant increase in circulating bile acids in septic shock patients with severe liver failure compared to healthy controls (61.8 µmol/L vs. 2.8 µmol/L, p = 0.0016). Circulating bile acids in these patients were capable to induce immunosuppression, as indicated by a significant increase in TGR5 activation by circulating bile acids (20.4% in severe liver failure vs. 2.8% in healthy controls, p = 0.0139).ConclusionsCirculating bile acids capable of inducing immunosuppression are present in septic shock patients with severe liver failure. Future studies should examine whether modulation of bile acid metabolism can improve the clinical course and outcome of sepsis in these patients.Graphical abstract

Amplified gut feelings under inflammation and depressed mood: A randomized fMRI trial on interoceptive pain in healthy volunteers

BackgroundInflammation and depressed mood constitute clinically relevant vulnerability factors for enhanced interoceptive sensitivity and chronic visceral pain, but their putative interaction remains untested in human mechanistic studies. We tested interaction effects of acute systemic inflammation and sad mood on the expectation and experience of visceral pain by combining experimental endotoxemia with a mood induction paradigm. MethodsThe double-blind, placebo-controlled, balanced crossover fMRI-trial in N = 39 healthy male and female volunteers involved 2 study days with either intravenous administration of low-dose lipopolysaccharide (LPS, 0.4 ng/kg body weight; inflammation condition) or saline (placebo condition). On each study, day two scanning sessions were conducted in an experimentally induced negative (i.e., sad) and in a neutral mood state, accomplished in balanced order. As a model of visceral pain, rectal distensions were implemented, which were initially calibrated to be moderately painful. In all sessions, an identical series of visceral pain stimuli was accomplished, signaled by predictive visual conditioning cues to assess pain anticipation. We assessed neural activation during the expectation and experience of visceral pain, along with unpleasantness ratings in a condition combining an inflammatory state with sad mood and in control conditions. All statistical analyses were accomplished using sex as covariate. ResultsLPS administration led to an acute systemic inflammatory response (inflammation X time interaction effects for TNF-α, IL-6, and sickness symptoms, all p <.001). The mood paradigm effectively induced distinct mood states (mood X time interaction, p <.001), with greater sadness in the negative mood conditions (both p <.001) but no difference between LPS and saline conditions. Significant main and interaction effects of inflammation and negative mood were observed for pain unpleasantness (all p <.05). During cued pain anticipation, a significant inflammation X mood interaction emerged for activation of the bilateral caudate nucleus and right hippocampus (all pFWE < 0.05). Main effects of both inflammation and mood were observed in multiple regions, including insula, midcingulate cortex, prefrontal gyri, and hippocampus for inflammation, and midcingulate, caudate, and thalamus for mood (all pFWE < 0.05). ConclusionsResults support an interplay of inflammation and sad mood on striatal and hippocampal circuitry engaged during visceral pain anticipation as well as on pain experience. This may reflect a nocebo mechanism, which may contribute to altered perception and interpretation of bodily signals. At the interface of affective neuroscience and the gut-brain axis, concurrent inflammation and negative mood may be vulnerability factors for chronic visceral pain.

Resolving sepsis-induced immunoparalysis via trained immunity by targeting interleukin-4 to myeloid cells

Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications.

Modulation of Pain Sensitivity by a Hyperventilatory Breathing Exercise and Cold Exposure Training.

Evidence indicates that healthy individuals who follow a training program comprised hyperventilatory breathing exercises and cold exposure can voluntarily activate their sympathetic nervous system and attenuate their systemic inflammatory response during experimental endotoxemia (intravenous administration of bacterial endotoxin). Furthermore, trained participants reported less endotoxemia-induced flu-like symptoms. However, it remained to be determined whether the effects on symptoms are due to the mitigated inflammatory response or involve direct analgesic effects of (elements of) the training program. In the present study, we used Nijmegen-Aalborg Screening Quantitative sensory testing (NASQ) to objectively map pain sensitivity using non-invasive stimuli to address this question. First, NASQ parameters were evaluated in 20 healthy volunteers before, during, and after the conduct of the hyperventilatory breathing exercise. Second, NASQ measurements were performed before and after 48 healthy volunteers followed different modalities of the training program: breathing exercise training, cold exposure training, the combination of both, or no training. Lastly, NASQ measurements were performed in these 48 subjects during experimental endotoxemia. Electrical pain detection thresholds increased during the breathing exercise (p = 0.001) as well as four hours afterwards (p = 0.03). Furthermore, cold exposure training resulted in lower VAS scores during hand immersion in ice water (p < 0.001). Systemic inflammation induced by administration of endotoxin nullified the decreased pain perception during the ice water test in subjects trained in cold exposure. A hyperventilatory breathing exercise decreases pain perception induced by an electrical stimulus. Furthermore, cold exposure training may decrease pain perception induced by hand immersion in ice water.

Atazanavir-induced unconjugated hyperbilirubinemia prevents vascular hyporeactivity during experimental human endotoxemia.

Inflammation-induced free radical release is important in the pathogenesis of several diseases, including atherosclerosis and sepsis. Heme oxygenase (HO) breaks down heme into carbon monoxide, iron, and biliverdin. Biliverdin IXα is directly converted to bilirubin by biliverdin reductase. Unconjugated bilirubin is a powerful antioxidant, and elevated levels have beneficial effects in preclinical models and human cardiovascular disease. However, its impact during acute inflammation in humans is unknown. In the present study, we investigated the impact of atazanavir-induced (unconjugated) hyperbilirubinemia on antioxidant capacity, inflammation, and vascular dysfunction in human experimental endotoxemia. Following double-blinded four-day treatment with atazanavir 2dd300 mg (or placebo), twenty healthy male volunteers received 2 ng/kg Escherichia coli lipopolysaccharide intravenously. Blood was drawn to determine the bilirubin levels, antioxidant capacity, and cytokine response. It was demonstrated that following atazanavir treatment, total bilirubin concentrations increased to maximum values of 4.67 (95%CI 3.91-5.59) compared to 0.82 (95%CI 0.64-1.07) mg/dL in the control group (p<0.01). Furthermore, the anti-oxidant capacity, as measured by the ferric-reducing ability of plasma (FRAP), was significantly increased with 36% in hyperbilirubinemia subjects (p<0.0001), and FRAP concentrations correlated strongly to bilirubin concentrations (R2=0.77, p<0.001). Hyperbilirubinemia attenuated the release of interleukin-10 from 377 (95%CI 233-609) to 219 (95%CI 152-318) pg/mL (p=0.01), whereas the release of pro-inflammatory cytokines remained unaltered. In vitro, in the absence of hyperbilirubinemia, atazanavir did not influence lipopolysaccharide-induced cytokine release in a whole blood assay. Vascular function was assessed using forearm venous occlusion plethysmography after intra-arterial infusion of acetylcholine and nitroglycerin. Hyperbilirubinemia completely prevented the LPS-associated blunted vascular response to acetylcholine and nitroglycerin. Atazanavir-induced hyperbilirubinemia increases antioxidant capacity, attenuates interleukin-10 release, and prevents vascular hyporesponsiveness during human systemic inflammation elicited by experimental endotoxemia. http://clinicaltrials.gov, identifier NCT00916448.