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Ghrelin alterations during experimental and human sepsis
Cytokine 127 (2020) 154937
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Cytokine journal homepage: www.elsevier.com/locate/cytokine
Ghrelin alterations during experimental and human sepsis a,1
b,1
b
a
T b
b
I. Nikitopoulou , E. Kampisiouli , E. Jahaj , A.G. Vassiliou , I. Dimopoulou , Z. Mastora , ⁎ S. Tsakirisa, K. Perreasa, M. Tzanelac, C. Routsib, S.E. Orfanosa,b,d, , A. Kotanidoua,b a
GP Livanos and M Simou Laboratories, 1st Department of Critical Care & Pulmonary Services, Medical School, National & Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece 1st Department of Critical Care & Pulmonary Services, Medical School, National & Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece c Department of Endocrinology, Diabetes and Metabolism, Evangelismos Hospital, Athens, Greece d 2nd Department of Critical Care, Medical School, National & Kapodistrian University of Athens, “Attikon” Hospital, Haidari, Athens, Greece b
A R T I C LE I N FO
A B S T R A C T
Keywords: Sepsis Ghrelin Endotoxemia Lipopolysaccharide
Background: Ghrelin is a hormone mainly produced by cells of the gastric mucosa, which has been shown to possess anti-inflammatory and immunomodulatory properties. The objective of the study was to investigate ghrelin levels during sepsis, as well as in an experimental sepsis model. Methods: All consecutive admissions to the ICU of a tertiary hospital in Athens, Greece were screened for eligibility during the study. Thirty four non-septic patients upon ICU admission who subsequently developed sepsis were enrolled. Clinical data and scores were recorded, and blood samples were obtained at baseline (upon ICU admission), and at sepsis development. Total and active ghrelin, leptin, and cytokines were measured. Moreover, lipopolysaccharide (LPS) was administered to mice in order to induce endotoxemia and at specified time points, blood and tissue samples were collected. Results: In patients, serum total and active ghrelin concentrations were significantly elevated in sepsis compared to baseline (553.8 ± 213.4 vs 193.5 ± 123.2, p < 0.001; 254.3 ± 70.6 vs 56.49 ± 16.3, p < 0.001). Active ghrelin levels at the sepsis stage were inversely correlated with SOFA score and length of stay in the ICU (p = 0.023 and p = 0.027 respectively). In the mouse endotoxemia model ghrelin levels were elevated following LPS treatment, and the same trend was observed for leptin, TNFα and IL-6. Ghrelin administration managed to reduce IL-6 levels in mouse serum and in BALF. Pulmonary expression of ghrelin and its receptor GHSR1a was found decreased in LPS-treated mice. Conclusions: In a well-defined cohort of ICU patients, we have demonstrated that active and total ghrelin increase in sepsis. The same is true for the experimental sepsis model used in the study. The inverse correlation of active ghrelin levels with SOFA score and length of ICU stay among septic patients is indicative of a potential protective role of active ghrelin during the septic process.
1. Introduction Sepsis, the major cause of mortality in the intensive care unit (ICU) worldwide [1], is a systemic inflammatory response leading eventually to multiorgan failure. During this immune response, pro-inflammatory cytokines are released and complex cellular and biochemical interactions take place. Management of sepsis is crucial; in addition to the excessive hospital costs, there is rising concern about the long term social implications following sepsis [2]. Ghrelin is a 28-amino acid acylated peptide produced mainly by stomach cells, which was found to be a ligand for the growth hormone
secretagogue receptor 1a (GHSR-1a) [3]. The active form of the peptide that binds and activates GHS-R1a requires a structural modification, namely attachment of n-octanoyl at the serine 3 residue [4]. Ghrelin and GHS-R1a are expressed in several tissues and cells of the immune system [5] and are involved in a variety of biological actions including metabolism, hemodynamics, but also inflammatory pathways [6,7]. Apart from endocrine functions, ghrelin has been reported to possess paracrine and autocrine functions, along with anti-inflammatory properties [8,9]. Exogenous administration of ghrelin has been proven to be immunomodulatory and anti-inflammatory [10]. Some previous studies have reported ghrelin levels during human
⁎
Corresponding author at: 2nd Department of Critical Care, Attikon University Hospital, 1, Rimini St; Haidari, Athens 12462, Greece. E-mail addresses: [email protected], [email protected] (S.E. Orfanos). 1 Equal contribution https://doi.org/10.1016/j.cyto.2019.154937 Received 25 January 2019; Received in revised form 24 October 2019; Accepted 19 November 2019 1043-4666/ © 2019 Elsevier Ltd. All rights reserved.
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endotoxemia or critical illness [11,12], but results seem to be contradictory. Endotoxin administration in healthy subjects was previously shown to initially augment and then decrease ghrelin levels [11]. Moreover, plasma ghrelin levels were reported higher in critically-ill patients compared to healthy volunteers in a study and lower in another [12,13]. It therefore appears that results in different studies are not consistent, most likely due to measurement of the acylated or desacylated isoforms and to the notable impact of sepsis stage on ghrelin expression. Since we have no knowledge concerning the effect of sepsis and its acute or chronic state on ghrelin expression, in this study we sought to detect serum ghrelin alterations in a cohort of ICU patients; in these initially non septic subjects that later developed sepsis, ghrelin isoforms were measured both at ICU admission in the absence of sepsis and at sepsis development. In addition to the role of endogenous ghrelin in this well-characterized group of critically-ill patients, we also investigated changes in ghrelin levels using a mouse model of endotoxemia.
Table 1 Data and complete diagnosis at ICU admission of the patients of ICU patients included in the study. Parameter
Patients
Number of patients
34
Sex Male Female Age (years) BMI (kg/m2) Pre-existing diabetes (n)
22 (64.7%) 12 (35.3%) 50 ± 3.3 27.4 ± 0.57 2 (5.8%)
Diagnosis (n) Surgical elective Neurosurgery Thoracic Abdominal Surgical emergency Neurosurgery Thoracic Abdominal Trauma Medical Cerebral hemorrhage Subarachnoid hemorrhage ARDS WBC Serum creatinine (mg/dl) Serum CRP (mg/dl) Serum PCT (ng/ml) Serum bilirubin (range) (mg/dl) APACHE II score SOFA score Mechanical Ventilation (days) LOS (days) ICU deaths n (%)
2. Materials and methods 2.1. Human study The protocol was approved by the Evangelismos Hospital Research Review Board - Ethics Committee (experimental study approval number 269/2011). All procedures were in accordance with the Helsinki Declaration and written consent was obtained from patients’ next of kin. Out of 178 overall admissions over the study period, 57 non septic subjects at ICU admission were found eligible to enter the study and finally 34 (22 male and 12 female) critically ill patients who subsequently developed sepsis were enrolled (Fig. 1). Inclusion criteria: nonsepsis upon ICU entry, sepsis development > 24 h post admission. Exclusion criteria were as follows: sepsis within the first 24 h of ICU admission according to Sepsis-3 criteria [14], BMI > 35 kg/m2 [15,16], age < 18 years, pregnancy, brain death, end-stage cancer, total ICU stay < 3 days, re-admission or transfer from another ICU, presence of infectious diseases (HIV, hepatitis) and oral intake of corticosteroids at an equivalent dosage of ≥1 mg/kg prednisone/day for a period of more than one month. The patients recruited in the study suffered from medical, surgical and trauma-related pathologies (Table 1). Following study enrolment, baseline (upon ICU admission) anthropometric data (height, weight) and detailed organ system-oriented medical history were recorded. The first blood draw was performed within the first 12 h post admission and the second within 12 h from sepsis diagnosis; all our patients initially developed the stage of sepsis [14]. Following whole blood collection in appropriate tubes (BD, New Jersey, USA) Pefabloc (Sigma-Aldrich, Germany) was added and samples were allowed to stand for 30 min at room temperature. Following
1 1
9 9
21 3 1 1 1 12,765 (5610–27,000) 0.78 ± 0.044 9 ± 1.6 0.726 ± 0.133 0.81 (0.18–2.9) 15 ± 1 7 ± 0.42 28 ± 3.5 34.2 ± 3.2 5 (14.7%)
Data are expressed as number of patients (n) and percentages of totals (%), except for age, duration of stay in ICU, APACHE and SOFA scores, and white blood cell count. BMI: body mass index; ARDS: acute respiratory distress syndrome; WBC: white blood cells; CRP: C-reactive protein; PCT: procalcitonin; APACHE: acute physiology and chronic health evaluation; SOFA: sequential organ failure assessment; LOS: length of stay; ICU = intensive care unit.
centrifugation at 2000g for 15 min, HCl was added to supernatant and samples were stored at −80 °C. Concentrations of total and active ghrelin, leptin, TNFα, IL-10 and IL-6 were determined by ELISA, according to manufacturer’s instructions (Millipore/Merck, Darmstadt, Germany and R&D Systems, Minnesota, USA respectively).
2.2. Animal study All experimentation was approved by the Evangelismos Hospital Research Review Board and the Veterinary Service of the governmental prefecture of Attica, Greece (approval protocol number K/1889/2011). The study was conducted in compliance with the European Union Directive 2010/63/EU and with the ARRIVE guidelines [17]. Animals were daily monitored and were randomly assigned into groups. All efforts were made to minimize animal distress and suffering. Mice were bred and maintained on a C57BL/6 background in the animal facilities of the “BSRC Alexander Fleming” (Athens, Greece) under specific pathogen–free conditions. Mice were then transported to the Animal Model Research Unit of Evangelismos Hospital where they were housed at 20–22 °C, with 55 ± 5% humidity, and a 12-hour lightdark cycle; food and water was given ad libitum during the experiment period. Eight week old wild type C57BL/6 mice were randomly caged in groups of five and were injected intraperitoneally with 20 mg/kg of lipopolysaccharide (LPS) prepared from Escherichia coli O111:B4 (Sigma-Aldrich, Germany). Control mice received sterile saline
Fig. 1. Flow diagram of the critically-ill patients included in the study. 2
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Fig. 2. (A) Serum levels of total ghrelin increase in ICU patients after sepsis development compared to the initial non-sepsis values. (B) Concentration of active ghrelin is significantly higher in serum of patients after sepsis development, as compared to non-sepsis levels. (C) Leptin levels in serum of critically ill patients, during non-sepsis and under sepsis. An increase is detected during the stage of sepsis. (D) Serum levels of anti-inflammatory cytokine IL-10 in our ICU patients’ cohort. Values are expressed as mean ± SEM. n = 34, * p < 0.05; *** p < 0.001.
Fig. 3. (A) Comparison of active ghrelin serum levels between critically-ill survivors and nonsurvivors. Active ghrelin was found significantly lower in non-survivors. (B) Association of sepsis patients’ disease severity with active ghrelin levels. SOFA score is inversely correlated with serum active ghrelin during sepsis. (C) The length of stay in the ICU is inversely correlated with serum active ghrelin (pg/ml) post sepsis development. Values are expressed as mean ± SEM (for (A)), * p < 0.05; ** p < 0.01.
Germany), AEBSF (Sigma-Aldrich, Germany) was immediately added to blood and HCl was then added to serum. For leptin, IL-6 and TNFα ELISA (R&D Systems, Minnesota, USA) procedures were performed according to manufacturer’s instructions. Total RNA was isolated from tissues using the PureLink RNA Mini kit (ThermoFisher Scientific, Massachusetts, USA), followed by cDNA synthesis using a PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio Inc., Shiga, Japan). PCR reaction mixtures were prepared using the KAPA SYBR Fast kit (Sigma-Aldrich), followed by quantitative PCR on a CFX Connect Real time PCR detection system (Bio-Rad Hercules, USA). The following primer pairs were used: human GHSR1, 5′-TCGTGGGT GCCTCGCT-3′ (forward) and 5′- CACCACTACAGCCAGCATTTTC-3′ (reverse); mouse GHSR1, 5′-TATGGGTGTCGAGCGTCTT-3′ (forward)
solution. At indicated time points 2 h, 4 h, 6 h and 16 h post injection mice were euthanized, blood was obtained from the abdominal inferior vena cava and bronchoalveolar lavage fluid (BALF) samples were collected via tracheotomy. The fluid was separated from cellular components by centrifugation at 1500 rpm for 10 min at 4 °C and supernatants were stored at −80 °C. Several tissues (stomach, intestine, lung, spleen, liver, kidney, adipose tissue) were snap-frozen in liquid nitrogen and stored at −80 °C or immersed in 4% buffered paraformaldehyde for histopathological evaluation. To test the effects of ghrelin administration, another group of mice was injected intraperitoneally with 10 μg of ghrelin (Phoenix Pharmaceuticals, St. Joseph, MO) 30 min prior to LPS administration. For total and active ghrelin ELISA (Millipore/Merck, Darmstadt, 3
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and 5′-GAGAATGGGGTTGATGGC-3′ (reverse); mouse GAPDH, 5′-AAC TTTGGCATTGTGGAAGG-3′ (forward) and 5′-ACACATTGGGGGTAGG AACA-3′ (reverse); human GAPDH, 5′-ATGGGGAAGGTGAAGGTCG-3′ (forward) and 5′- GGGGTCATTGATGGCAACAATA-3′ (reverse); mouse ghrelin, 5′-AGCATGCTCTGGATGGACATG-3′ (forward) and 5′-AGGCC TGTCCGTGGTTACTTGT-3′ (reverse). GAPDH served as the internal control to normalize the amount of loaded cDNA.
Table 2 Characteristics of survivor and non-survivor populations included in the study. Parameter
Survivors
Non-survivors
Number of patients
29
5
Sex Male Female Age (years) BMI (kg/m2)
19 10 48 ± 3.6 27.4 ± 0.59
3 2 59 ± 9.1 27.6 ± 1.94
Diagnosis (n) Surgical elective Neurosurgery Thoracic Abdominal Surgical emergency Neurosurgery Thoracic Abdominal Trauma Medical Cerebral hemorrhage Subarachnoid hemorrhage ARDS WBC Serum creatinine (mg/dl) Serum CRP (mg/dl) Serum PCT (ng/ml) APACHE II score SOFA score Mechanical Ventilation (days) LOS (days)
2.3. Statistical analysis Data are presented as means ± SEM and as medians with interquartile range as appropriate. Comparisons of mice (saline and LPS) groups were made using the Mann-Whitney test, while among human measurements the Wilcoxon rank test was used. Multiple comparisons were assessed by ANOVA using the post hoc Kruskal-Wallis test. Correlations were analyzed by the Pearson correlation test. Values of p < 0.05 were considered statistically significant.
1 1
8 8
1 1
17 3 1 1 1 13,747 0.78 ± 0.05 9.248 ± 1.89 0.642 ± 0.126 14.3 ± 1.034 6.84 ± 0.44 24.8 ± 3.3 32.3 ± 3.09
4 3 1 1 1 11,814 0.75 ± 0.063 7.82 ± 3.34 1.208 ± 0.521 18 ± 2.16 7 ± 1.45 44.8 ± 12.5 44.8 ± 12.52
3. Results 3.1. Demographics of patients The flow diagram of patients’ enrolment is presented in Fig. 1, while the baseline characteristics of the patients included in the study are summarized in Table 1. Data on patients’ diagnosis, length of stay in the ICU and severity are provided in Table 1. Overall, 34 ICU patients aged 50 ± 3.3 years, with a BMI of 27.4 ± 0.6 kg/m2 were recruited at Evangelismos Hospital.
BMI: body mass index; ARDS: acute respiratory distress syndrome; WBC: white blood cells; CRP: C-reactive protein; PCT: procalcitonin; APACHE: acute physiology and chronic health evaluation; SOFA: sequential organ failure assessment; LOS: length of stay; ICU = intensive care unit.
3.2. Ghrelin levels in critically-ill patients Total and active ghrelin levels were measured repetitively in sera of ICU patients, ≤12 h post admission as well as at sepsis development. As shown in Fig. 2A, total ghrelin levels at sepsis were found elevated compared to baseline (i.e. non-sepsis) values (553.8 ± 213.4 vs Fig. 4. Effect of endotoxemia on serum ghrelin levels in mice. (A) Blood was withdrawn 2, 4, 6 and 16 h post LPS injection, serum total ghrelin levels were measured by ELISA and were shown to increase in response to LPS. (B) Changes in circulating active ghrelin levels during the course of endotoxemia. Active ghrelin levels in serum were measured 2, 4, 6 and 16 h post LPS injection. (C) Serum mouse leptin responses to intraperitoneal endotoxin. At indicated time points, leptin levels in mouse serum were determined and an increase in leptin over time is noticed. Values are expressed as medians (box edges: 25–75 centiles, whiskers: minimum to maximum values), n = 5 mice per group. * p < 0.05; ** p < 0.01.
4
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Fig. 5. Alterations in levels of pro- and anti-inflammatory cytokines during different time points of mouse endotoxemia. (A) Serum TNFα levels in mice increase after LPS challenge, as measured by ELISA. (B) TNFα levels in BALF of LPS-treated mice tend to elevate compared to those of saline-treated mice. (C) Effect of endotoxemia on IL-6 production. Levels of IL-6 in serum of LPS-injected mice are significantly higher than in corresponding control groups. (D) IL-6 levels in mouse BALF. BALF was collected from mice and IL-6 levels were determined by ELISA, showing an increase upon endotoxemia. (E) Effect of endotoxemia on serum IL-10 levels compared to corresponding control groups. (F) IL-10 levels in mice BALF were determined by ELISA, showing a decrease upon endotoxemia. Values are expressed as means ± SEM. * p < 0.05; *** p < 0.001.
We thus sought to look for possible correlations between ghrelin and other variables crucial in sepsis. As depicted in Fig. 3B and C, active ghrelin values at sepsis development were inversely correlated with sequential organ failure assessment (SOFA) score (r = −0.4, p = 0.023), as well as with the duration of stay in the ICU (r = −0.385, p = 0.027). In order to delineate possible mechanisms involving ghrelin in sepsis progression, the expression of GHSR1α in our ICU patients was investigated. GHSR1α mRNA levels were detected in patients’ blood, and an increasing trend was noticed during sepsis, but the difference between sepsis and non-sepsis stages did not reach statistical significance (data not shown).
193.5 ± 123.2 pg/ml, p < 0.001). The same increasing pattern was shown for active ghrelin levels (Fig. 2B, 254.3 ± 70.6 vs 56.49 ± 16.3 pg/ml, p < 0.001). 3.3. Serum leptin and cytokine levels in critically-ill patients Leptin levels in these patients were measured and were found higher in sepsis than at baseline (22.1 ± 7.2 vs. 35.9 ± 11.2 ng/ml, p = 0.009, Fig. 2C). Similar results were obtained for the proinflammatory cytokine IL-6; levels were increased in sepsis when compared to baseline values (data not shown). Interestingly, levels of the anti-inflammatory cytokine IL-10 drop in sepsis compared to non-sepsis without though reaching statistical significance (Fig. 2D).
3.5. Serum ghrelin and leptin levels during endotoxemia in mice 3.4. Correlations of serum active ghrelin with sepsis parameters Total ghrelin levels in serum of mice treated with LPS were elevated, compared to serum levels of control mice (Fig. 4A). This increase was noticed within 2 h after endotoxin administration and remained elevated until the 16 h time point. Active ghrelin levels in serum of mice treated with LPS seemed to
Notably, active ghrelin levels were significantly higher in survivors than in non-survivors and this finding was observed at both stages, sepsis and non-sepsis (Fig. 3A). Parameters and indices related to survivors vs non-survivors are provided in Table 2. 5
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Fig. 6. Effect of exogenous ghrelin administration on IL-6 and IL-10 levels during endotoxemia in mice. (A) Mouse serum IL-6 levels increase after LPS challenge and decrease upon ghrelin administration at 16 h. (B) IL-6 levels in BALF of mice receiving exogenous ghrelin prior to LPS are lower compared to those of LPS-treated mice. (C) In mice injected with ghrelin, IL-10 serum levels increase 2 h after LPS administration. Values are expressed as means ± SEM. * p < 0.05; ** p < 0.01.
levels in serum were reduced; the greater and statistically significant decrease was observed 16 h post LPS administration. Similarly, ghrelin administration significantly reduced BALF IL-6 levels, 2 h and 16 h after LPS (Fig. 6B). Finally, as indicated in Fig. 6C, treatment with ghrelin increased serum IL-10 levels compared to levels of LPS-injected mice.
follow that increasing trend 2, 4, and 6 h after the intraperitoneal injection compared to their respective control animals, without reaching statistical significance (Fig. 4B). At the 16 h time point post LPS, the opposite phenomenon was observed; active ghrelin levels decreased in the LPS group when compared to the saline group. The highest levels of total ghrelin were measured in serum of LPSchallenged mice 6 h after the injection, while those of active ghrelin were found in mice 2 h post LPS. Additionally, serum leptin levels increased in response to LPS starting 2 h post injection and reached a maximum at 16 h (Fig. 4C).
3.8. Expression of ghrelin and GHSR1α in mouse tissues Ghrelin mRNA was detected in tissues of mice of the experimental groups, with the stomach and lung showing the highest expression (Fig. 7A). Expression of ghrelin in LPS lungs showed a significant decrease compared to saline, while in kidney and small intestine from LPS-challenged mice it was found increased (Fig. 7B). Concerning GHSR1α expression, a similar pattern was observed; the receptor’s mRNA was significantly increased in the intestine and kidney of LPSchallenged mice compared to control mice (Fig. 7C). On the contrary, expression of GHSR1α in lungs following LPS injection showed a decrease over saline-treated mouse lungs.
3.6. Effects of endotoxin on pro-inflammatory and anti-inflammatory cytokines in mice Circulating TNFα increased from baseline values at all time points after endotoxin injection (Fig. 5A). Similarly, levels of circulating IL-6 in LPS-challenged mice showed a great increase compared to undetectable levels of saline-treated animals and remained elevated until 16 h post injection (Fig. 5C). The aforementioned proinflammatory molecules were also determined in the BALF of all experimental groups (Fig. 5B and D). As shown in Fig. 5D, IL-6 was the cytokine exhibiting greater differences in BALF between saline and LPS treated mice. Levels of IL-10 were measured at all time points in mouse serum and BALF. Notably, IL-10 concentration was undetectable or low in serum 2, 4 and 6 h post LPS, but significantly increased 16 h after LPS. At the same time, levels of IL-10 in BALF of LPS-treated mice followed a decreasing trend until the time point of 16 h (Fig. 5E and F).
4. Discussion In the present study, serum ghrelin alterations in a cohort of initially non septic ICU patients were studied; repeated measurements of total and active ghrelin were performed at admission in the ICU, as well as at sepsis development (definitions according to [14]). Levels of total ghrelin and of the acylated form of ghrelin which is considered biologically active (i.e. active ghrelin) were found elevated in sepsis compared to baseline, depicting the role of endogenous ghrelin in sepsis pathophysiology. In addition, leptin levels were found increased at sepsis development and were accompanied by the anticipated elevation of IL-6 levels [18] and decrease of IL-10 levels. We showed that levels of serum acylated ghrelin levels at ICU admission and at sepsis development were significantly higher in surviving sepsis patients. Interestingly, serum active ghrelin levels at sepsis development are inversely
3.7. Effect of ghrelin administration on cytokine concentration during mouse endotoxemia In order to explore how ghrelin is connected with the inflammatory effects observed, exogenous ghrelin was administered to mice. As shown in Fig. 6A, when septic mice were treated with ghrelin, IL-6 6
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An older study focusing on postoperative intra-abdominal sepsis, also revealed a ghrelin increase in plasma [20], and serum leptin concentration in patients with acute sepsis was shown to increase compared to healthy controls [21]. Leptin, a key cytokine in food intake regulation that possesses a modulatory role in inflammatory process, was previously reported to increase upon endotoxin injection [22]. We also noticed a prolonged elevation of mouse serum leptin after endotoxin, in accordance with data previously shown in dogs [23]. Under normal conditions ghrelin and leptin exert opposing effects, thus the observed simultaneous elevations of both molecules in our study could be attributed to endotoxemia, as previously discussed [23]. Upon LPS challenge immune cell stimulation may also result in subsequent ghrelin production [24,25]. Thus, a regulatory signaling role in the immune system is suggested for ghrelin, affecting cytokine and leptin expression [25]. Although it has been previously shown that ghrelin levels decrease in the plasma of rats following cecal ligation and puncture (CLP) [26], other studies have reported elevated levels in a rat model of endotoxic shock [24], during endotoxemia-induced acute kidney injury [27], and in plasma of endotoxin-challenged dogs [23]. In our experimental setting, ghrelin levels increase in mouse serum after LPS injection. In this regard, plasma ghrelin elevation during endotoxin shock has been previously justified as an adaptive mechanism of protection [24]. Ghrelin clearance drops remarkably in late sepsis and this could be attributed to renal or hepatic dysfunctions taking place during the septic process [28]. In our endotoxemia model a beneficial effect has been shown in animals receiving exogenous ghrelin and this is further supported by the literature: intravenous ghrelin administration in a rat sepsis model reduced lung proinflammatory cytokine production and neutrophil infiltration, while it improved survival [29]. Similarly, beneficial effects have been shown in bleomycin-induced acute lung injury (ALI), pancreatitis-induced ALI [30,31] and monocrotaline-induced pulmonary hypertension [32]. The observed decrease in lung ghrelin expression following LPS is in agreement with a previous study showing lower pulmonary protein levels of ghrelin after CLP in rats [29] and could be related to the increased bacterial load and lung injury due to LPS. Besides, other studies previously revealed a role for ghrelin in fetal lung development [33] and recognized GHSR1a binding sites in lung parenchyma [34]. Pulmonary ghrelin seems to be of particular interest, since lung complications appear to be the most challenging in intensive care units. Renal ghrelin expression in our mouse model was found higher after LPS challenge, and the same applied for GHSR1a expression, supporting previous data on acute kidney injury [27]. As for expression in the small intestine, to our knowledge this is the first study reporting elevated ghrelin and ghrelin receptor mRNA expression after treatment of mice with endotoxin. Sepsis and endotoxemia are multifactorial phenomena; still ghrelin appears to possess properties to attenuate inflammatory responses and thus be potentially useful during these processes.
Fig. 7. Ghrelin and ghrelin receptor GHSR1a expression during mouse endotoxemia. (A) Relative mRNA expression levels of ghrelin in various mouse tissues. As expected, highest expression is noticed in stomach, which serves as the positive control. (B) Alterations in pulmonary, renal and intestinal levels of ghrelin after LPS. Relative ghrelin mRNA expression drops in lung but it increases in kidney and intestine of LPS-treated mice. (C) GHSR1a mRNA levels are higher in kidney and intestine of LPS-treated mice as compared to control mice (saline), while pulmonary levels are found relatively lower. Values were normalized to GAPDH (endogenous reference gene). * p < 0.05; ** p < 0.01.
5. Conclusion
associated with SOFA score and with the duration of ICU stay. In the present study serum ghrelin and leptin levels were also examined in a murine endotoxemia model and were found increased at every time point studied after exposure to LPS. TNFα and IL-6 levels were elevated both in serum and BALF of endotoxin-treated mice. Pulmonary ghrelin expression was decreased in lungs of mice treated with endotoxin, compared to saline-treated controls. Administration of exogenous ghrelin managed to decrease IL-6 concentration in mouse serum and BALF and to increase IL-10 levels in serum. Ghrelin has been shown to possess therapeutic potential, due to its anti-inflammatory properties [19]. Regulatory mechanisms in the progression of sepsis are complex, but it seems likely that elevation of circulating ghrelin can be beneficial in critical illness.
Conclusively, our study highlights a role for ghrelin in sepsis pathophysiology. On one hand, endogenous ghrelin production seems to be affected by endotoxin and by the various biological disruptions observed in the septic process, although the exact mechanisms of ghrelin regulation remain to be fully delineated. On the other hand, we found a ghrelin-dependent suppression of IL-6 levels and simultaneous increase of IL-10 levels after sepsis induction, a finding indicative of an effective immune response related to ghrelin. The multiple biological effects of ghrelin are by now accepted, as well as the wide tissue distribution of ghrelin and its functional receptor [35]. Ghrelin has been proven to possess anti-inflammatory effects and our study extends these findings by suggesting a beneficial and 7
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potentially protective role of ghrelin in critical illness. [15]
Source of funding
[16]
This study was supported by the nonprofit institute ‘THORAX’ Research Center for Intensive and Emergency Thoracic Medicine, and by a grant from GlaxoSmithKline Greece (to SEO, grant number 3000584938).
[17]
[18]
Declaration of Competing Interest [19]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[20]
[21]
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Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Ghrelin alterations during experimental and human sepsis a,1
b,1
b
a
T b
b
I. Nikitopoulou , E. Kampisiouli , E. Jahaj , A.G. Vassiliou , I. Dimopoulou , Z. Mastora , ⁎ S. Tsakirisa, K. Perreasa, M. Tzanelac, C. Routsib, S.E. Orfanosa,b,d, , A. Kotanidoua,b a
GP Livanos and M Simou Laboratories, 1st Department of Critical Care & Pulmonary Services, Medical School, National & Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece 1st Department of Critical Care & Pulmonary Services, Medical School, National & Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece c Department of Endocrinology, Diabetes and Metabolism, Evangelismos Hospital, Athens, Greece d 2nd Department of Critical Care, Medical School, National & Kapodistrian University of Athens, “Attikon” Hospital, Haidari, Athens, Greece b
A R T I C LE I N FO
A B S T R A C T
Keywords: Sepsis Ghrelin Endotoxemia Lipopolysaccharide
Background: Ghrelin is a hormone mainly produced by cells of the gastric mucosa, which has been shown to possess anti-inflammatory and immunomodulatory properties. The objective of the study was to investigate ghrelin levels during sepsis, as well as in an experimental sepsis model. Methods: All consecutive admissions to the ICU of a tertiary hospital in Athens, Greece were screened for eligibility during the study. Thirty four non-septic patients upon ICU admission who subsequently developed sepsis were enrolled. Clinical data and scores were recorded, and blood samples were obtained at baseline (upon ICU admission), and at sepsis development. Total and active ghrelin, leptin, and cytokines were measured. Moreover, lipopolysaccharide (LPS) was administered to mice in order to induce endotoxemia and at specified time points, blood and tissue samples were collected. Results: In patients, serum total and active ghrelin concentrations were significantly elevated in sepsis compared to baseline (553.8 ± 213.4 vs 193.5 ± 123.2, p < 0.001; 254.3 ± 70.6 vs 56.49 ± 16.3, p < 0.001). Active ghrelin levels at the sepsis stage were inversely correlated with SOFA score and length of stay in the ICU (p = 0.023 and p = 0.027 respectively). In the mouse endotoxemia model ghrelin levels were elevated following LPS treatment, and the same trend was observed for leptin, TNFα and IL-6. Ghrelin administration managed to reduce IL-6 levels in mouse serum and in BALF. Pulmonary expression of ghrelin and its receptor GHSR1a was found decreased in LPS-treated mice. Conclusions: In a well-defined cohort of ICU patients, we have demonstrated that active and total ghrelin increase in sepsis. The same is true for the experimental sepsis model used in the study. The inverse correlation of active ghrelin levels with SOFA score and length of ICU stay among septic patients is indicative of a potential protective role of active ghrelin during the septic process.
1. Introduction Sepsis, the major cause of mortality in the intensive care unit (ICU) worldwide [1], is a systemic inflammatory response leading eventually to multiorgan failure. During this immune response, pro-inflammatory cytokines are released and complex cellular and biochemical interactions take place. Management of sepsis is crucial; in addition to the excessive hospital costs, there is rising concern about the long term social implications following sepsis [2]. Ghrelin is a 28-amino acid acylated peptide produced mainly by stomach cells, which was found to be a ligand for the growth hormone
secretagogue receptor 1a (GHSR-1a) [3]. The active form of the peptide that binds and activates GHS-R1a requires a structural modification, namely attachment of n-octanoyl at the serine 3 residue [4]. Ghrelin and GHS-R1a are expressed in several tissues and cells of the immune system [5] and are involved in a variety of biological actions including metabolism, hemodynamics, but also inflammatory pathways [6,7]. Apart from endocrine functions, ghrelin has been reported to possess paracrine and autocrine functions, along with anti-inflammatory properties [8,9]. Exogenous administration of ghrelin has been proven to be immunomodulatory and anti-inflammatory [10]. Some previous studies have reported ghrelin levels during human
⁎
Corresponding author at: 2nd Department of Critical Care, Attikon University Hospital, 1, Rimini St; Haidari, Athens 12462, Greece. E-mail addresses: [email protected], [email protected] (S.E. Orfanos). 1 Equal contribution https://doi.org/10.1016/j.cyto.2019.154937 Received 25 January 2019; Received in revised form 24 October 2019; Accepted 19 November 2019 1043-4666/ © 2019 Elsevier Ltd. All rights reserved.
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endotoxemia or critical illness [11,12], but results seem to be contradictory. Endotoxin administration in healthy subjects was previously shown to initially augment and then decrease ghrelin levels [11]. Moreover, plasma ghrelin levels were reported higher in critically-ill patients compared to healthy volunteers in a study and lower in another [12,13]. It therefore appears that results in different studies are not consistent, most likely due to measurement of the acylated or desacylated isoforms and to the notable impact of sepsis stage on ghrelin expression. Since we have no knowledge concerning the effect of sepsis and its acute or chronic state on ghrelin expression, in this study we sought to detect serum ghrelin alterations in a cohort of ICU patients; in these initially non septic subjects that later developed sepsis, ghrelin isoforms were measured both at ICU admission in the absence of sepsis and at sepsis development. In addition to the role of endogenous ghrelin in this well-characterized group of critically-ill patients, we also investigated changes in ghrelin levels using a mouse model of endotoxemia.
Table 1 Data and complete diagnosis at ICU admission of the patients of ICU patients included in the study. Parameter
Patients
Number of patients
34
Sex Male Female Age (years) BMI (kg/m2) Pre-existing diabetes (n)
22 (64.7%) 12 (35.3%) 50 ± 3.3 27.4 ± 0.57 2 (5.8%)
Diagnosis (n) Surgical elective Neurosurgery Thoracic Abdominal Surgical emergency Neurosurgery Thoracic Abdominal Trauma Medical Cerebral hemorrhage Subarachnoid hemorrhage ARDS WBC Serum creatinine (mg/dl) Serum CRP (mg/dl) Serum PCT (ng/ml) Serum bilirubin (range) (mg/dl) APACHE II score SOFA score Mechanical Ventilation (days) LOS (days) ICU deaths n (%)
2. Materials and methods 2.1. Human study The protocol was approved by the Evangelismos Hospital Research Review Board - Ethics Committee (experimental study approval number 269/2011). All procedures were in accordance with the Helsinki Declaration and written consent was obtained from patients’ next of kin. Out of 178 overall admissions over the study period, 57 non septic subjects at ICU admission were found eligible to enter the study and finally 34 (22 male and 12 female) critically ill patients who subsequently developed sepsis were enrolled (Fig. 1). Inclusion criteria: nonsepsis upon ICU entry, sepsis development > 24 h post admission. Exclusion criteria were as follows: sepsis within the first 24 h of ICU admission according to Sepsis-3 criteria [14], BMI > 35 kg/m2 [15,16], age < 18 years, pregnancy, brain death, end-stage cancer, total ICU stay < 3 days, re-admission or transfer from another ICU, presence of infectious diseases (HIV, hepatitis) and oral intake of corticosteroids at an equivalent dosage of ≥1 mg/kg prednisone/day for a period of more than one month. The patients recruited in the study suffered from medical, surgical and trauma-related pathologies (Table 1). Following study enrolment, baseline (upon ICU admission) anthropometric data (height, weight) and detailed organ system-oriented medical history were recorded. The first blood draw was performed within the first 12 h post admission and the second within 12 h from sepsis diagnosis; all our patients initially developed the stage of sepsis [14]. Following whole blood collection in appropriate tubes (BD, New Jersey, USA) Pefabloc (Sigma-Aldrich, Germany) was added and samples were allowed to stand for 30 min at room temperature. Following
1 1
9 9
21 3 1 1 1 12,765 (5610–27,000) 0.78 ± 0.044 9 ± 1.6 0.726 ± 0.133 0.81 (0.18–2.9) 15 ± 1 7 ± 0.42 28 ± 3.5 34.2 ± 3.2 5 (14.7%)
Data are expressed as number of patients (n) and percentages of totals (%), except for age, duration of stay in ICU, APACHE and SOFA scores, and white blood cell count. BMI: body mass index; ARDS: acute respiratory distress syndrome; WBC: white blood cells; CRP: C-reactive protein; PCT: procalcitonin; APACHE: acute physiology and chronic health evaluation; SOFA: sequential organ failure assessment; LOS: length of stay; ICU = intensive care unit.
centrifugation at 2000g for 15 min, HCl was added to supernatant and samples were stored at −80 °C. Concentrations of total and active ghrelin, leptin, TNFα, IL-10 and IL-6 were determined by ELISA, according to manufacturer’s instructions (Millipore/Merck, Darmstadt, Germany and R&D Systems, Minnesota, USA respectively).
2.2. Animal study All experimentation was approved by the Evangelismos Hospital Research Review Board and the Veterinary Service of the governmental prefecture of Attica, Greece (approval protocol number K/1889/2011). The study was conducted in compliance with the European Union Directive 2010/63/EU and with the ARRIVE guidelines [17]. Animals were daily monitored and were randomly assigned into groups. All efforts were made to minimize animal distress and suffering. Mice were bred and maintained on a C57BL/6 background in the animal facilities of the “BSRC Alexander Fleming” (Athens, Greece) under specific pathogen–free conditions. Mice were then transported to the Animal Model Research Unit of Evangelismos Hospital where they were housed at 20–22 °C, with 55 ± 5% humidity, and a 12-hour lightdark cycle; food and water was given ad libitum during the experiment period. Eight week old wild type C57BL/6 mice were randomly caged in groups of five and were injected intraperitoneally with 20 mg/kg of lipopolysaccharide (LPS) prepared from Escherichia coli O111:B4 (Sigma-Aldrich, Germany). Control mice received sterile saline
Fig. 1. Flow diagram of the critically-ill patients included in the study. 2
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Fig. 2. (A) Serum levels of total ghrelin increase in ICU patients after sepsis development compared to the initial non-sepsis values. (B) Concentration of active ghrelin is significantly higher in serum of patients after sepsis development, as compared to non-sepsis levels. (C) Leptin levels in serum of critically ill patients, during non-sepsis and under sepsis. An increase is detected during the stage of sepsis. (D) Serum levels of anti-inflammatory cytokine IL-10 in our ICU patients’ cohort. Values are expressed as mean ± SEM. n = 34, * p < 0.05; *** p < 0.001.
Fig. 3. (A) Comparison of active ghrelin serum levels between critically-ill survivors and nonsurvivors. Active ghrelin was found significantly lower in non-survivors. (B) Association of sepsis patients’ disease severity with active ghrelin levels. SOFA score is inversely correlated with serum active ghrelin during sepsis. (C) The length of stay in the ICU is inversely correlated with serum active ghrelin (pg/ml) post sepsis development. Values are expressed as mean ± SEM (for (A)), * p < 0.05; ** p < 0.01.
Germany), AEBSF (Sigma-Aldrich, Germany) was immediately added to blood and HCl was then added to serum. For leptin, IL-6 and TNFα ELISA (R&D Systems, Minnesota, USA) procedures were performed according to manufacturer’s instructions. Total RNA was isolated from tissues using the PureLink RNA Mini kit (ThermoFisher Scientific, Massachusetts, USA), followed by cDNA synthesis using a PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio Inc., Shiga, Japan). PCR reaction mixtures were prepared using the KAPA SYBR Fast kit (Sigma-Aldrich), followed by quantitative PCR on a CFX Connect Real time PCR detection system (Bio-Rad Hercules, USA). The following primer pairs were used: human GHSR1, 5′-TCGTGGGT GCCTCGCT-3′ (forward) and 5′- CACCACTACAGCCAGCATTTTC-3′ (reverse); mouse GHSR1, 5′-TATGGGTGTCGAGCGTCTT-3′ (forward)
solution. At indicated time points 2 h, 4 h, 6 h and 16 h post injection mice were euthanized, blood was obtained from the abdominal inferior vena cava and bronchoalveolar lavage fluid (BALF) samples were collected via tracheotomy. The fluid was separated from cellular components by centrifugation at 1500 rpm for 10 min at 4 °C and supernatants were stored at −80 °C. Several tissues (stomach, intestine, lung, spleen, liver, kidney, adipose tissue) were snap-frozen in liquid nitrogen and stored at −80 °C or immersed in 4% buffered paraformaldehyde for histopathological evaluation. To test the effects of ghrelin administration, another group of mice was injected intraperitoneally with 10 μg of ghrelin (Phoenix Pharmaceuticals, St. Joseph, MO) 30 min prior to LPS administration. For total and active ghrelin ELISA (Millipore/Merck, Darmstadt, 3
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and 5′-GAGAATGGGGTTGATGGC-3′ (reverse); mouse GAPDH, 5′-AAC TTTGGCATTGTGGAAGG-3′ (forward) and 5′-ACACATTGGGGGTAGG AACA-3′ (reverse); human GAPDH, 5′-ATGGGGAAGGTGAAGGTCG-3′ (forward) and 5′- GGGGTCATTGATGGCAACAATA-3′ (reverse); mouse ghrelin, 5′-AGCATGCTCTGGATGGACATG-3′ (forward) and 5′-AGGCC TGTCCGTGGTTACTTGT-3′ (reverse). GAPDH served as the internal control to normalize the amount of loaded cDNA.
Table 2 Characteristics of survivor and non-survivor populations included in the study. Parameter
Survivors
Non-survivors
Number of patients
29
5
Sex Male Female Age (years) BMI (kg/m2)
19 10 48 ± 3.6 27.4 ± 0.59
3 2 59 ± 9.1 27.6 ± 1.94
Diagnosis (n) Surgical elective Neurosurgery Thoracic Abdominal Surgical emergency Neurosurgery Thoracic Abdominal Trauma Medical Cerebral hemorrhage Subarachnoid hemorrhage ARDS WBC Serum creatinine (mg/dl) Serum CRP (mg/dl) Serum PCT (ng/ml) APACHE II score SOFA score Mechanical Ventilation (days) LOS (days)
2.3. Statistical analysis Data are presented as means ± SEM and as medians with interquartile range as appropriate. Comparisons of mice (saline and LPS) groups were made using the Mann-Whitney test, while among human measurements the Wilcoxon rank test was used. Multiple comparisons were assessed by ANOVA using the post hoc Kruskal-Wallis test. Correlations were analyzed by the Pearson correlation test. Values of p < 0.05 were considered statistically significant.
1 1
8 8
1 1
17 3 1 1 1 13,747 0.78 ± 0.05 9.248 ± 1.89 0.642 ± 0.126 14.3 ± 1.034 6.84 ± 0.44 24.8 ± 3.3 32.3 ± 3.09
4 3 1 1 1 11,814 0.75 ± 0.063 7.82 ± 3.34 1.208 ± 0.521 18 ± 2.16 7 ± 1.45 44.8 ± 12.5 44.8 ± 12.52
3. Results 3.1. Demographics of patients The flow diagram of patients’ enrolment is presented in Fig. 1, while the baseline characteristics of the patients included in the study are summarized in Table 1. Data on patients’ diagnosis, length of stay in the ICU and severity are provided in Table 1. Overall, 34 ICU patients aged 50 ± 3.3 years, with a BMI of 27.4 ± 0.6 kg/m2 were recruited at Evangelismos Hospital.
BMI: body mass index; ARDS: acute respiratory distress syndrome; WBC: white blood cells; CRP: C-reactive protein; PCT: procalcitonin; APACHE: acute physiology and chronic health evaluation; SOFA: sequential organ failure assessment; LOS: length of stay; ICU = intensive care unit.
3.2. Ghrelin levels in critically-ill patients Total and active ghrelin levels were measured repetitively in sera of ICU patients, ≤12 h post admission as well as at sepsis development. As shown in Fig. 2A, total ghrelin levels at sepsis were found elevated compared to baseline (i.e. non-sepsis) values (553.8 ± 213.4 vs Fig. 4. Effect of endotoxemia on serum ghrelin levels in mice. (A) Blood was withdrawn 2, 4, 6 and 16 h post LPS injection, serum total ghrelin levels were measured by ELISA and were shown to increase in response to LPS. (B) Changes in circulating active ghrelin levels during the course of endotoxemia. Active ghrelin levels in serum were measured 2, 4, 6 and 16 h post LPS injection. (C) Serum mouse leptin responses to intraperitoneal endotoxin. At indicated time points, leptin levels in mouse serum were determined and an increase in leptin over time is noticed. Values are expressed as medians (box edges: 25–75 centiles, whiskers: minimum to maximum values), n = 5 mice per group. * p < 0.05; ** p < 0.01.
4
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Fig. 5. Alterations in levels of pro- and anti-inflammatory cytokines during different time points of mouse endotoxemia. (A) Serum TNFα levels in mice increase after LPS challenge, as measured by ELISA. (B) TNFα levels in BALF of LPS-treated mice tend to elevate compared to those of saline-treated mice. (C) Effect of endotoxemia on IL-6 production. Levels of IL-6 in serum of LPS-injected mice are significantly higher than in corresponding control groups. (D) IL-6 levels in mouse BALF. BALF was collected from mice and IL-6 levels were determined by ELISA, showing an increase upon endotoxemia. (E) Effect of endotoxemia on serum IL-10 levels compared to corresponding control groups. (F) IL-10 levels in mice BALF were determined by ELISA, showing a decrease upon endotoxemia. Values are expressed as means ± SEM. * p < 0.05; *** p < 0.001.
We thus sought to look for possible correlations between ghrelin and other variables crucial in sepsis. As depicted in Fig. 3B and C, active ghrelin values at sepsis development were inversely correlated with sequential organ failure assessment (SOFA) score (r = −0.4, p = 0.023), as well as with the duration of stay in the ICU (r = −0.385, p = 0.027). In order to delineate possible mechanisms involving ghrelin in sepsis progression, the expression of GHSR1α in our ICU patients was investigated. GHSR1α mRNA levels were detected in patients’ blood, and an increasing trend was noticed during sepsis, but the difference between sepsis and non-sepsis stages did not reach statistical significance (data not shown).
193.5 ± 123.2 pg/ml, p < 0.001). The same increasing pattern was shown for active ghrelin levels (Fig. 2B, 254.3 ± 70.6 vs 56.49 ± 16.3 pg/ml, p < 0.001). 3.3. Serum leptin and cytokine levels in critically-ill patients Leptin levels in these patients were measured and were found higher in sepsis than at baseline (22.1 ± 7.2 vs. 35.9 ± 11.2 ng/ml, p = 0.009, Fig. 2C). Similar results were obtained for the proinflammatory cytokine IL-6; levels were increased in sepsis when compared to baseline values (data not shown). Interestingly, levels of the anti-inflammatory cytokine IL-10 drop in sepsis compared to non-sepsis without though reaching statistical significance (Fig. 2D).
3.5. Serum ghrelin and leptin levels during endotoxemia in mice 3.4. Correlations of serum active ghrelin with sepsis parameters Total ghrelin levels in serum of mice treated with LPS were elevated, compared to serum levels of control mice (Fig. 4A). This increase was noticed within 2 h after endotoxin administration and remained elevated until the 16 h time point. Active ghrelin levels in serum of mice treated with LPS seemed to
Notably, active ghrelin levels were significantly higher in survivors than in non-survivors and this finding was observed at both stages, sepsis and non-sepsis (Fig. 3A). Parameters and indices related to survivors vs non-survivors are provided in Table 2. 5
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Fig. 6. Effect of exogenous ghrelin administration on IL-6 and IL-10 levels during endotoxemia in mice. (A) Mouse serum IL-6 levels increase after LPS challenge and decrease upon ghrelin administration at 16 h. (B) IL-6 levels in BALF of mice receiving exogenous ghrelin prior to LPS are lower compared to those of LPS-treated mice. (C) In mice injected with ghrelin, IL-10 serum levels increase 2 h after LPS administration. Values are expressed as means ± SEM. * p < 0.05; ** p < 0.01.
levels in serum were reduced; the greater and statistically significant decrease was observed 16 h post LPS administration. Similarly, ghrelin administration significantly reduced BALF IL-6 levels, 2 h and 16 h after LPS (Fig. 6B). Finally, as indicated in Fig. 6C, treatment with ghrelin increased serum IL-10 levels compared to levels of LPS-injected mice.
follow that increasing trend 2, 4, and 6 h after the intraperitoneal injection compared to their respective control animals, without reaching statistical significance (Fig. 4B). At the 16 h time point post LPS, the opposite phenomenon was observed; active ghrelin levels decreased in the LPS group when compared to the saline group. The highest levels of total ghrelin were measured in serum of LPSchallenged mice 6 h after the injection, while those of active ghrelin were found in mice 2 h post LPS. Additionally, serum leptin levels increased in response to LPS starting 2 h post injection and reached a maximum at 16 h (Fig. 4C).
3.8. Expression of ghrelin and GHSR1α in mouse tissues Ghrelin mRNA was detected in tissues of mice of the experimental groups, with the stomach and lung showing the highest expression (Fig. 7A). Expression of ghrelin in LPS lungs showed a significant decrease compared to saline, while in kidney and small intestine from LPS-challenged mice it was found increased (Fig. 7B). Concerning GHSR1α expression, a similar pattern was observed; the receptor’s mRNA was significantly increased in the intestine and kidney of LPSchallenged mice compared to control mice (Fig. 7C). On the contrary, expression of GHSR1α in lungs following LPS injection showed a decrease over saline-treated mouse lungs.
3.6. Effects of endotoxin on pro-inflammatory and anti-inflammatory cytokines in mice Circulating TNFα increased from baseline values at all time points after endotoxin injection (Fig. 5A). Similarly, levels of circulating IL-6 in LPS-challenged mice showed a great increase compared to undetectable levels of saline-treated animals and remained elevated until 16 h post injection (Fig. 5C). The aforementioned proinflammatory molecules were also determined in the BALF of all experimental groups (Fig. 5B and D). As shown in Fig. 5D, IL-6 was the cytokine exhibiting greater differences in BALF between saline and LPS treated mice. Levels of IL-10 were measured at all time points in mouse serum and BALF. Notably, IL-10 concentration was undetectable or low in serum 2, 4 and 6 h post LPS, but significantly increased 16 h after LPS. At the same time, levels of IL-10 in BALF of LPS-treated mice followed a decreasing trend until the time point of 16 h (Fig. 5E and F).
4. Discussion In the present study, serum ghrelin alterations in a cohort of initially non septic ICU patients were studied; repeated measurements of total and active ghrelin were performed at admission in the ICU, as well as at sepsis development (definitions according to [14]). Levels of total ghrelin and of the acylated form of ghrelin which is considered biologically active (i.e. active ghrelin) were found elevated in sepsis compared to baseline, depicting the role of endogenous ghrelin in sepsis pathophysiology. In addition, leptin levels were found increased at sepsis development and were accompanied by the anticipated elevation of IL-6 levels [18] and decrease of IL-10 levels. We showed that levels of serum acylated ghrelin levels at ICU admission and at sepsis development were significantly higher in surviving sepsis patients. Interestingly, serum active ghrelin levels at sepsis development are inversely
3.7. Effect of ghrelin administration on cytokine concentration during mouse endotoxemia In order to explore how ghrelin is connected with the inflammatory effects observed, exogenous ghrelin was administered to mice. As shown in Fig. 6A, when septic mice were treated with ghrelin, IL-6 6
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An older study focusing on postoperative intra-abdominal sepsis, also revealed a ghrelin increase in plasma [20], and serum leptin concentration in patients with acute sepsis was shown to increase compared to healthy controls [21]. Leptin, a key cytokine in food intake regulation that possesses a modulatory role in inflammatory process, was previously reported to increase upon endotoxin injection [22]. We also noticed a prolonged elevation of mouse serum leptin after endotoxin, in accordance with data previously shown in dogs [23]. Under normal conditions ghrelin and leptin exert opposing effects, thus the observed simultaneous elevations of both molecules in our study could be attributed to endotoxemia, as previously discussed [23]. Upon LPS challenge immune cell stimulation may also result in subsequent ghrelin production [24,25]. Thus, a regulatory signaling role in the immune system is suggested for ghrelin, affecting cytokine and leptin expression [25]. Although it has been previously shown that ghrelin levels decrease in the plasma of rats following cecal ligation and puncture (CLP) [26], other studies have reported elevated levels in a rat model of endotoxic shock [24], during endotoxemia-induced acute kidney injury [27], and in plasma of endotoxin-challenged dogs [23]. In our experimental setting, ghrelin levels increase in mouse serum after LPS injection. In this regard, plasma ghrelin elevation during endotoxin shock has been previously justified as an adaptive mechanism of protection [24]. Ghrelin clearance drops remarkably in late sepsis and this could be attributed to renal or hepatic dysfunctions taking place during the septic process [28]. In our endotoxemia model a beneficial effect has been shown in animals receiving exogenous ghrelin and this is further supported by the literature: intravenous ghrelin administration in a rat sepsis model reduced lung proinflammatory cytokine production and neutrophil infiltration, while it improved survival [29]. Similarly, beneficial effects have been shown in bleomycin-induced acute lung injury (ALI), pancreatitis-induced ALI [30,31] and monocrotaline-induced pulmonary hypertension [32]. The observed decrease in lung ghrelin expression following LPS is in agreement with a previous study showing lower pulmonary protein levels of ghrelin after CLP in rats [29] and could be related to the increased bacterial load and lung injury due to LPS. Besides, other studies previously revealed a role for ghrelin in fetal lung development [33] and recognized GHSR1a binding sites in lung parenchyma [34]. Pulmonary ghrelin seems to be of particular interest, since lung complications appear to be the most challenging in intensive care units. Renal ghrelin expression in our mouse model was found higher after LPS challenge, and the same applied for GHSR1a expression, supporting previous data on acute kidney injury [27]. As for expression in the small intestine, to our knowledge this is the first study reporting elevated ghrelin and ghrelin receptor mRNA expression after treatment of mice with endotoxin. Sepsis and endotoxemia are multifactorial phenomena; still ghrelin appears to possess properties to attenuate inflammatory responses and thus be potentially useful during these processes.
Fig. 7. Ghrelin and ghrelin receptor GHSR1a expression during mouse endotoxemia. (A) Relative mRNA expression levels of ghrelin in various mouse tissues. As expected, highest expression is noticed in stomach, which serves as the positive control. (B) Alterations in pulmonary, renal and intestinal levels of ghrelin after LPS. Relative ghrelin mRNA expression drops in lung but it increases in kidney and intestine of LPS-treated mice. (C) GHSR1a mRNA levels are higher in kidney and intestine of LPS-treated mice as compared to control mice (saline), while pulmonary levels are found relatively lower. Values were normalized to GAPDH (endogenous reference gene). * p < 0.05; ** p < 0.01.
5. Conclusion
associated with SOFA score and with the duration of ICU stay. In the present study serum ghrelin and leptin levels were also examined in a murine endotoxemia model and were found increased at every time point studied after exposure to LPS. TNFα and IL-6 levels were elevated both in serum and BALF of endotoxin-treated mice. Pulmonary ghrelin expression was decreased in lungs of mice treated with endotoxin, compared to saline-treated controls. Administration of exogenous ghrelin managed to decrease IL-6 concentration in mouse serum and BALF and to increase IL-10 levels in serum. Ghrelin has been shown to possess therapeutic potential, due to its anti-inflammatory properties [19]. Regulatory mechanisms in the progression of sepsis are complex, but it seems likely that elevation of circulating ghrelin can be beneficial in critical illness.
Conclusively, our study highlights a role for ghrelin in sepsis pathophysiology. On one hand, endogenous ghrelin production seems to be affected by endotoxin and by the various biological disruptions observed in the septic process, although the exact mechanisms of ghrelin regulation remain to be fully delineated. On the other hand, we found a ghrelin-dependent suppression of IL-6 levels and simultaneous increase of IL-10 levels after sepsis induction, a finding indicative of an effective immune response related to ghrelin. The multiple biological effects of ghrelin are by now accepted, as well as the wide tissue distribution of ghrelin and its functional receptor [35]. Ghrelin has been proven to possess anti-inflammatory effects and our study extends these findings by suggesting a beneficial and 7
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potentially protective role of ghrelin in critical illness. [15]
Source of funding
[16]
This study was supported by the nonprofit institute ‘THORAX’ Research Center for Intensive and Emergency Thoracic Medicine, and by a grant from GlaxoSmithKline Greece (to SEO, grant number 3000584938).
[17]
[18]
Declaration of Competing Interest [19]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[20]
[21]
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