LOW DOSE OF ESMOLOL ATTENUATES SEPSIS-INDUCED IMMUNOSUPPRESSION VIA MODULATING T-LYMPHOCYTE APOPTOSIS AND DIFFERENTIATION

ABSTRACT Background: Immunosuppression caused by immune cell apoptosis and an imbalance of T helper 2 cells (TH2) and T helper 1 cells (TH1), is associated with poor outcomes in septic patients. Esmolol was reported to improve survival by modulating immune responses in septic shock. Whether esmolol could alleviate sepsis-induced immunosuppression and the optimal dose are unclear. Methods: Four hours after cecal ligation and puncture (CLP), Wistar rats were randomized into CLP, CLP + E-5 (esmolol: 5 mg·kg−1·h−1) and CLP + E-18 (esmolol: 18 mg·kg−1·h−1) groups. Eight rats were underwent sham operation. Eighteen hours after CLP, hemodynamics and organ histological injuries were evaluated, peripheral blood mononuclear cells apoptosis and T-lymphocyte subsets counts were determined by flow cytometry, and the expression of p-Akt, Bcl-2, cleaved Caspase-3, and p-Erk1/2 in splenic CD4+ T-lymphocytes was determined by western blot and immunohistochemistry. β1-Adrenoreceptor expressions were evaluated using real-time polymerase chain reaction and immunohistochemistry. Results: Cecal ligation and puncture induced tachycardia, hypotension, hyperlactatemia, and multiple organ injury. Heart rate was unchanged in the CLP + E-5 group but decreased in the CLP + E-18 group. Hypotension, lactatemia, and multiple organ injuries were improved only in the CLP + E-5 group. T-lymphocyte apoptosis and TH2/TH1 ratio was decreased in CLP + E-5 but not in CLP + E-18. p-Akt and Bcl-2 expressions were increased, while cleaved Caspase-3 and p-Erk1/2 expressions were decreased in CLP + E-5. β1-Adrenoreceptor expressions were unchanged in both CLP + E-5 and CLP + E-18 groups. Conclusions: Low dose of esmolol reduced T-lymphocyte apoptosis and restored TH2/TH1 ratio in septic shock. Esmolol might modulate Akt/Bcl-2/Caspase-3 pathway to relieve T-lymphocyte apoptosis and inhibit Erk1/2 activity to decrease TH0 differentiation to TH2. Esmolol may be a potential immunoregulator of septic shock.


INTRODUCTION
Septic shock develops as a dysregulated host inflammatory response to infection, resulting in multiple organ dysfunction that is associated with high mortality worldwide (1). Both pro-and anti-inflammatory immune responses occur after the onset of sepsis. If sepsis persists, patients will enter a markedly immunosuppressive state (2). Immunosuppressed septic patients are at a high risk of secondary nosocomial infection resulting in an increasing 13% mortality of septic patients (3). Immune cell apoptosis, such as circulating monocytes, B-lymphocytes and T-lymphocytes, and upregulated T helper 2 cells (T H 2)/T helper 1 cells (T H 1) ratio are the most reported characteristics contributing to immunosuppression during septic shock (4,5).
In experimental and clinical studies, esmolol, a highly selective ultrashort-acting β 1 -adrenoreceptor blocker, was recently reported to improve cardiovascular function and survival in septic shock (6)(7)(8). The beneficial effects of esmolol in septic shock have been previously considered because of its hemodynamic effects (9). However, recent evidence has shown that the beneficial effects of esmolol in septic shock are associated with immunomodulation (10,11). However, how esmolol influences the immune response in septic shock and the optimal dose are unclear.
Lymphocytes and monocytes expressed β 1 -adrenoreceptors (12), which belong to the G-protein-coupled receptor (GPCR) super family. The GPCRs couple to a G protein heterotrimer including the α, β, and γ subunits in the intracellular region (Fig. 1). G protein α (Gα) binds to the G protein βγ dimer (Gβγ) in the inactive state. The GPCR activation leads to the dissociation of Gα and Gβγ. Gβγ modulates protein kinase B (Akt) via phosphatidylinositide 3-kinases (PI3K) (13). Akt activation leads to B-cell leukemia/lymphoma 2-associated death promoter (Bad) phosphorylation, resulting in B-cell leukemia/lymphoma 2 (Bcl-2) release, ultimately promoting cell survival (14). Our previous study showed that blocking the β 1 -adrenoreceptor by esmolol could increase Akt phosphorylation in cardiovascular tissue (10). Qi et al. reported that Akt phosphorylation decreased sepsis-induced cardiomyocyte apoptosis via upregulation of Bcl-2 and downregulation of cleaved Caspase-3 both in vitro and in vivo (15). Thus, we aimed to determine whether esmolol could reduce sepsis-induced peripheral blood mononuclear cell (PBMC) apoptosis by modulating Akt/Bcl-2/Caspase-3 pathway. Activated Gα induces extracellular regulated protein kinases (Erk) phosphorylation through the adenylyl cyclase-cyclic adenosine monophosphate-protein kinase A pathway (16). Furthermore, Erk is an obligatory mediator of the T H 2 differentiation pathway (17). Interleukin 4, endogenously produced upon T-cell receptor (TCR) cross-linking, establishes a positive feedback loop through IL-4R that further reinforces IL-4 expression in T H 0 (18,19), inducing T H 0 differentiation to T H 2. Erk promotes T H 2 differentiation by activating the early phase of TCR-dependent IL-4 production (17). Hence, we also aimed to determine whether esmolol could reduce T H 0 differentiation to T H 2 by inhibiting Erk1/2 activation.

Animals
Adult male Wistar rats weighing 300-400 g were obtained from the Center for Animal Experiments of Wuhan University. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Animal Experiment Center of Wuhan University (E2020072901) and followed the institutional and national guidelines.

Study design
The cecal ligation and puncture (CLP) model was used to develop a septic shock model as previously described (20). Four hours after CLP, all rats were randomized into three groups (CLP [n = 8], CLP + E-5 [CLP with esmolol infused at 5 mg·kg −1 ·h −1 , n = 8], CLP + E-18 [CLP with esmolol infused at 18 mg·kg −1 ·h −1 , n = 8]). Eight rats were under sham operation. Four hours after surgery, all rats received fluid resuscitation (Saline 10 mL·kg −1 ·h −1 ), antibiotic (meropenem 10 mg·kg −1 ), and analgesic (nalbuphine 0.2 mg·kg −1 ·h −1 ) for 14 hours. The rats in the CLP + E-5 and CLP + E-18 groups received the infusion of esmolol initiated 4 hours after surgery for a period of 14 hours. Assessments were performed 18 hours after the CLP or sham surgery. Eighteen-hour mortality was collected in each group.

Dose selection of esmolol
A high dose with heart rate reduction and a low dose without heart rate reduction were chosen in the study to determine the optimal dose. Infusing septic shock rats with esmolol at 18 kg −1 ·h −1 induced heart rate reduction compared with CLP group in the previous study (10). To compare with the previous study, 18 mg·kg −1 ·h −1 was chosen as the high dose with heart rate reduction in the study. The low dose was chosen according to our previous study (10). In this study, we did a dose gradient analysis (esmolol infused at 1, 5, and 18 mg·kg −1 ·h −1 ) and found that esmolol infused at both 5 and 1 mg·kg −1 ·h −1 did not reduce heart rate but 5 mg·kg −1 ·h −1 presented better immunomodulatory effects compared with 1 mg·kg −1 ·h −1 . Thus, esmolol infused at 5 mg·kg −1 ·h −1 was chosen as the low dose without heart rate reduction.

Hemodynamics and organ injuries measurement
The pressure transducer catheter was inserted into the right carotid artery of anesthetized rats and connected to a BL-420N biological signal recorder (Taimeng, Chengdu, China) to measure the HR and MAP. Arterial blood was collected for lactate detection using an ABL800 FLEX blood-gas Analyzer (Radiometer, Denmark). Heart, lung, and spleen tissue sections were stained with hematoxylin and eosin, and morphological changes were observed under the optical microscope (Olympus, Tokyo, Japan) at 200Â magnification. Histopathological lesions were quantified using five randomly selected fields per slide (21).

Cytokine analysis
The level of IL-4 in the plasma was measured using a rat IL-4 enzyme-linked immunosorbent assay kit (Bioswamp, Wuhan, China) according to the manufacturer's protocols. The results are expressed as picograms of the measured cytokine per milliliter of plasma.

Quantitative real-time polymerase chain reaction
Total RNA of splenic CD4 + T-lymphocytes from six rats in each group was extracted. According to the manufacturer's manual, the purified mRNA from each sample was reverse transcribed into complementary deoxyribonucleic acid (cDNA) using PrimeScript RT Master Mix (Vazyme, Nanjing, China)(Supplemental Table S2, http://links.lww.com/SHK/B646). Quantitative real-time polymerase chain reactions (qRT-PCR) was performed using the UltraSYBR Mixture (CWbio, Beijing, China). The relative mRNA expression level of the β 1 -adrenoreceptor in splenic CD4 + T-lymphocytes was calculated using the 2 −ΔΔCt method.

Statistical analysis
Data are expressed as median with interquartile range (IQR) in main text and tables and as median with upper edges of error bars representing the 75th percentile in figures. The Mann-Whitney test was performed to evaluate the differences between the sham and CLP groups. The Kruskal-Wallis test was performed between the CLP, CLP + E-5, and CLP + E-18 groups. When the Kruskal-Wallis test was significant at the 5% level, Dunnett multiple post hoc comparisons were performed. The data were plotted using GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA) and analyzed using IBM-SPSS Statistics 23.0 (IBM Corp, NY).  Table 1). The CLP induced (1) cardiac muscle fibers destruction, congestion, and inflammatory infiltration (Supplemental Fig. S4A, http://links. lww.com/SHK/B646); (2) apparent inflammatory cells aggregation, intra-alveolar capillary hemorrhages, and thickening of the alveolar walls in lung tissues (Supplemental Fig. S4B, http:// links.lww.com/SHK/B646); and (3) depletion of reticuloendothelial cells and lymphocytes in spleen tissues (Supplemental Fig.  S4C, http://links.lww.com/SHK/B646). The heart ( P = 0.002), lung ( P = 0.002), and spleen ( P = 0.002) injury scores were increased in the CLP group ( Table 1).

Effects of different doses of esmolol on T H 0 differentiation
The CLP induced an increase in both T H 1( P = 0.005) and T H 2 ( P = 0.001) levels compared with the sham group ( Fig. 3A

Effect of different doses of esmolol on circulatory IL-4 level
Compared with the sham group, CLP was associated with increased plasma levels of IL-4 ( P<0.001)(Supplemental Fig. S7, http://links.lww.com/SHK/B646). Addition of esmolol at 5 mg·kg −1 ·h −1 in CLP rats resulted in a decrease in plasma IL-4 level ( P = 0.031). However, there were no significant difference in plasma IL-4 level between the CLP and CLP + E-18 groups ( P = 0.837).

Effects of different doses of esmolol on the β 1 -adrenoreceptor expression on T-lymphocytes
Compared with the sham group, CLP decreased mRNA ( P = 0.002) and protein ( P = 0.029) expression of β 1adrenoreceptor in splenic CD4 + T-lymphocytes tested by qRT-PCR and immunohistochemistry ( Fig. 5 and Supplemental Figure S8

DISCUSSION
The main result of the study is that blocking β 1 -adrenoreceptors by esmolol decreased circulating T-lymphocyte apoptosis and restored peripheral blood T H 2/T H 1 ratio in septic shock model. Akt/Bcl-2/Caspase-3 pathway, which was associated with T-lymphocyte apoptosis, was found to be modulated by esmolol. Erk1/2 activity, which promoted T H 0 differentiation to T H 2, was revealed to be inhibited by esmolol. Esmolol at low dose without heart rate reduction showed better immunomodulatory effects than at high dose with heart rate reduction.

Model characteristics
In the study, the CLP model was used to establish the septic shock model. All rats were resuscitated with adapted fluids. Antibiotics was infused 4 hours after CLP to mimic clinical settings. As in previous studies (9,10,20,23), all rats that underwent CLP showed the typical characteristics of septic shock, including hypotension, hyperlactatemia, and multiple organ injuries, including heart, lung, and spleen (Table 1 and Supplemental Fig. S4, http:// links.lww.com/SHK/B646).

Effects of esmolol on survival
Ackland et al. (24) pretreated sepsis rats with β 1 -adrenoreceptor blockers (metoprolol and atenolol) 2 days before injection of LPS improved survival. Metoprolol increased median time to death in sepsis rats when pretreated 2 days before CLP. However, both metoprolol and atenolol failed to improve survival when treatment commenced 6 hours after induction of sepsis in their study. The doses used of metoprolol and atenolol resulted in a 20% reduction in the heart rate from baseline in the study. Medical treatment is usually after sepsis insult in clinical settings. To reproduce clinical setting of septic patients, the following studies commenced treatment after sepsis insult. Mori et al. (25) administrated septic rats with esmolol infusion 1 hour after CLP. The dose of esmolol also reduced heart rate by approximately 20% as compared with baseline. They found the survival time was significantly improved in esmolol group. Kimmoun et al. (9) infused septic rats with esmolol 4 hours after CLP with a dose reducing heart rate as compared with CLP group. Median time to death was also increased in esmolol-treated septic rats. In contrast to previous studies, Ibrahim-zada et al. (26) infused sepsis rats with esmolol 4 hours after injection of LPS with a very low dose without any effect on myocardial function and also showed survival improvement in esmolol group. Previous results showed that both high and low doses of β 1 -adrenoreceptor blockers improved survival in experimental sepsis. In our study, the 18-hour mortality was 33.3%, 11.1%, and 20%, respectively, in CLP, CLP + E-5, and CLP + E-18 group, which was consistent with previous studies (9,(24)(25)(26).

Effects of esmolol on T-lymphocyte apoptosis
Previous studies have reported that immunosuppression predominately results from apoptosis of monocytes, B-lymphocytes, and T-lymphocytes in septic shock patients (3,27). In our study, CLP induced apoptosis of circulating monocytes, B-lymphocytes, and T-lymphocytes, which was similar to the clinical settings. Overstimulation of immune cells via adrenergic receptors by catecholamines, which are secreted by the sympathetic nervous system overactivation, contributes to their apoptosis in septic shock (28,29). Our results showed that blocking β 1 -adrenergic receptors by esmolol at low dose significantly reduced T-lymphocyte apoptosis in rats with septic shock.
Mechanisms of esmolol on T-lymphocyte apoptosis β 1 -Adrenoreceptor could modulate Akt via Gβγ/PI3K (13). Our previous study showed that blocking β 1 -adrenoreceptors by esmolol could increase Akt phosphorylation (10). Akt activation leads to Bad phosphorylation resulting in Bcl-2 release, ultimately promoting cell survival (14). Our results revealed that low dose of esmolol increased Akt phosphorylation and Bcl-2 expression and reduced cleaved Caspase-3 in splenic CD4 + T-lymphocytes in septic shock models. The results were consistent with the report by Qi et al. (15) in cardiomyocytes that phosphorylation of Akt induced upregulation of Bcl-2 and downregulation of cleaved Caspase-3. Therefore, esmolol might reduce T-lymphocytes apoptosis in septic shock by modulating the Akt/Bcl-2/Caspase-3 pathway (Fig. 1).

Effects of esmolol on T H 2/T H 1 ratio
Previous studies have shown an imbalance of T-lymphocyte subpopulations in septic patients, such as an augmented T H 2/ T H 1 ratio (5,30,31). Our results showed an increased peripheral blood T H 2/T H 1 ratio in septic shock models as clinical settings. Infusion of esmolol at low dose restored the ratio of T H 2 and T H 1 in septic shock.

Mechanisms of esmolol on T H 2 differentiation
Induction of T H 0 into the T H 2 differentiation pathway depends to a significant extent on IL-4 produced upon TCR cross-linking (32). Interleukin 4 establishes a positive feedback loop through IL-4R, which further reinforces IL-4 expression while silencing the IFN-γ locus at the same time in T H 0 (18,19). Erk influences TCR-dependent activation of IL-4 gene transcription through association to the proximal promoter. Thus, Erk modulates T H 0 differentiation via TCR-dependent IL-4 production. Activation of the β 1 -adrenoreceptor could induce Erk phosphorylation through the adenylyl cyclase-cyclic adenosine monophosphateprotein kinase A pathway (16). Our results showed that blocking β 1 -adrenoreceptor by esmolol decreased CLP-induced Erk1/ 2 phosphorylation. Circulating IL-4 level was also decreased in esmolol-treated group, which was consistent with the study by Manon et al. (11). In their study, blocking β 1 -adrenoreceptor by esmolol decreased circulating IL-4 level in septic shock mice.
These findings supported that esmolol might reduce T H 0 differentiation to T H 2 in septic shock by decreasing IL-4 production via inhibiting of Erk1/2 activation (Fig. 1).

Different doses of esmolol on immunomodulation
The optimal dose of esmolol for immunoregulation of septic shock remains unclear. Our results showed that esmolol at low dose without heart rate reduction rather than at high dose with heart rate reduction significantly decreased T-lymphocytes apoptosis and restored T H 2/T H 1 ratio in septic shock models. Thus, low dose of esmolol might be more promising for modulating the immune response in septic shock. More studies are needed to confirm our findings.

Effects of esmolol on β 1 -adrenoreceptor on T-lymphocytes
In septic shock, excessive stimulation by catecholamine resulted in the reduction of β 1 -adrenoreceptor density in cardiomyocytes, which was restored by esmolol treatment (6). Our results demonstrated that the mRNA and protein expression of β 1 -adrenoreceptor also decreased in T-lymphocytes in septic shock rats. However, esmolol infusion at different doses did not restore the mRNA or protein expression of β 1 -adrenoreceptor on T-lymphocytes in septic shock rats. The effects of esmolol on its receptor expression may vary in different cells. Further study is needed to elucidate the mechanisms.

Study limitation
In fact, esmolol also decreased peripheral blood T H 0 differentiation to T H 1 in septic shock in the study. However, the mechanisms were not explored in the study. Our work is just a starting point for investigating the effects and mechanisms of esmolol on T H 0 differentiation. More follow-up work is needed. Besides, the immune status varies during the course of septic shock. The optimal time to the initiation of esmolol treatment requires future investigation. Lastly, only male rats were used in this study. Female rats should be used in the following researches to complete date for the entire population.

CONCLUSIONS
Esmolol reduced circulating T-lymphocyte apoptosis and restored the peripheral blood T H 2/T H 1 ratio. Esmolol might modulate the Akt/Bcl-2/Caspase-3 pathway to relieve T-lymphocyte apoptosis and inhibit Erk1/2 activity to decrease peripheral blood T H 0 differentiation to T H 2. Esmolol at low dose without heart rate reduction showed better immunomodulatory effects than at high dose. Esmolol at low dose may be a potential immunoregulator of septic shock.