Effects of propofol and its formulation components on macrophages and neutrophils in obese and lean animals

Abstract We hypothesized whether propofol or active propofol component (2,6‐diisopropylphenol [DIPPH] and lipid excipient [LIP‐EXC]) separately may alter inflammatory mediators expressed by macrophages and neutrophils in lean and obese rats. Male Wistar rats (n = 10) were randomly assigned to receive a standard (lean) or obesity‐inducing diet (obese) for 12 weeks. Animals were euthanized, and alveolar macrophages and neutrophils from lean and obese animals were exposed to propofol (50 µM), active propofol component (50 µM, 2,6‐DIPPH), and lipid excipient (soybean oil, purified egg phospholipid, and glycerol) for 1 h. The primary outcome was IL‐6 expression after propofol and its components exposure by alveolar macrophages extracted from bronchoalveolar lavage fluid. The secondary outcomes were the production of mediators released by macrophages from adipose tissue, and neutrophils from lung and adipose tissues, and neutrophil migration. IL‐6 increased after the exposure to both propofol (median [interquartile range] 4.14[1.95–5.20]; p = .04) and its active component (2,6‐DIPPH) (4.09[1.67–5.91]; p = .04) in alveolar macrophages from obese animals. However, only 2,6‐DIPPH increased IL‐10 expression (7.59[6.28–12.95]; p = .001) in adipose tissue‐derived macrophages. Additionally, 2,6‐DIPPH increased C‐X‐C chemokine receptor 2 and 4 (CXCR2 and CXCR4, respectively) in lung (10.08[8.23–29.01]; p = .02; 1.55[1.49–3.43]; p = .02) and adipose tissues (8.78[4.15–11.57]; p = .03; 2.86[2.17–3.71]; p = .01), as well as improved lung‐derived neutrophil migration (28.00[−3.42 to 45.07]; p = .001). In obesity, the active component of propofol affected both the M1 and M2 markers as well as neutrophils in both alveolar and adipose tissue cells, suggesting that lipid excipient may hinder the effects of active propofol.


| INTRODUC TI ON
Obesity is a complex multifactorial disease associated with several comorbidities. 1 The chronic low-grade inflammatory condition of obesity orchestrated by immune cell disruption has relevant negative consequences. 2,3 Anesthesia and the perioperative stress response can modulate several immunological reactions, which are very diverse and still not fully understood in obesity. The immunomodulatory effects of anesthetic agents have been observed in clinical 4,5 and preclinical 6,7 studies involving the perioperative period and models of acute inflammation that were distinct from the low-grade systemic inflammation in obesity.
Propofol, a widely used anesthetic agent, is composed of its active component (2,) and lipid excipient (LIP-EXC) (soybean oil, glycerol, and egg lecithin), which may have different biological activities. 8 Propofol immunomodulatory properties have been investigated in several models of inflammation, and that might have had an impact on perioperative outcomes, depending on the context, favoring pro-or anti-inflammatory responses. In vitro studies, propofol decreases chemotaxis, phagocytic activities, oxidative ability, 9 and the release of inflammatory mediators 10 by reducing nuclear factor (NF)-κB activation. 11 In neutrophils, propofol composition is also capable of inhibiting elastase release. 12 Recently, in experimental obesity, controversial results that may be associated with the composition of propofol yielded increased airway resistance and interleukin (IL)-6 levels. 13 These results have led to questions regarding the mechanisms of propofol composition or its main formulation components in obesityrelated inflammation. Within this context, we hypothesized that both active propofol components, DIPPH and LIP-EXC, may have different impacts on inflammatory mediators expressed by macrophages, neutrophils, and other structural cells in obesity. The primary outcome was to assess changes in IL-6 expression induced by propofol and its components in alveolar macrophages extracted from bronchoalveolar lavage fluid. The secondary outcomes were to analyze the production of mediators from lung tissue, neutrophil migration, mediators associated with vascular adhesion in endothelial cells, and fibrogenesis in fibroblasts.
Moreover, mediators released by macrophages and neutrophils were also evaluated in adipose tissue.

| Study approval
This study was approved by the Ethics Committee of the Health Sciences Center of Federal University of Rio de Janeiro (020/16 CEUA-UFRJ Rio de Janeiro, Brazil). All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the US National Academy of Sciences. Animals experiments followed the ARRIVE guidelines. 14 Non-specific-pathogen-free animals were housed in a controlled temperature (23°C) and controlled light-dark cycle (12-12 h), with free access to water and food, with acclimation for 1 week.

| Bronchoalveolar lavage
Alveolar macrophages were collected from lungs by bronchoalveolar lavage. A polyethylene cannula was inserted into the trachea and a total volume of 1.5 ml of warm phosphate-buffered saline (PBS) containing 10 mM ethylenediamine tetraacetic acid (EDTA) at 37°C was instilled and the lungs washed three times. Samples were centrifuged at 300× g for 5 min at 4°C. The supernatant was removed, and cells in the pellet were resuspended in 1× PBS.
To ensure enough cells for assay, alveolar macrophages from each group were pooled and pressed through a 40μm mesh filter into a single-cell suspension. Cells were then cultured in a six-well culture plate at 37°C with 5% CO 2 at a concentration of 10 5 cells per well in 2 ml of Roswell Park Memorial Institute 1640 medium (Sigma Chemical Co) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 2 mM L-glutamine (Life Technologies), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. 15 After incubation for 2 h, non-adherent cells were washed off with PBS, and the medium was replaced.

| Isolation of lung cells
Lungs were removed and the left lung was used to isolate neutrophils, endothelial cells, and fibroblasts.

K E Y W O R D S
adipose tissue, inflammation, lung, macrophages, neutrophils, obesity, propofol 2.4.1 | Lung neutrophils Lung neutrophils were isolated according to the standard method of sedimentation, centrifugation in Hystopaque density gradient separation followed by hypotonic lysis of red blood cells. 12 The purified neutrophils were immediately submitted to drug exposure.

| Lung endothelial cells
The left lung was cut into small pieces and digested with col- penicillin/streptomycin, and 1% glutamine (Invitrogen Life Technologies), and transferred to six-well plates pre-coated with 0.1% gelatin. 16

| Lung fibroblasts
Lung tissue was cut into small pieces and subjected to enzymatic collagenase digestion for 45-60 min at 37°C. The reaction was blocked by adding Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and immediately centrifuged at 300× g for 5 min. Cells were plated in DMEM-F12, 10% FBS, 1000 U/ml penicillin/streptomycin antibiotic solution, and 2 mM L-glutamine and kept in a humidified chamber with 5% CO 2 at 37°C.

| Isolation of macrophages and neutrophils in adipose tissue
Epididymal fat pad tissue was collected, rinsed in PBS, transferred to a Petri dish, and cut into small pieces. The dissected pieces (around 0.2-0.8 cm 3 ) were washed with PBS, cut into smaller fragments (1 mm), and subsequently digested with collagenase type I (1 mg/ ml) with agitation for 40 min at 37°C. Proteolysis was stopped with supplemented media, centrifuged, the pellet was resuspended, and then macrophages and neutrophils were isolated through a Percoll gradient solution. 17

| Cell culture
Macrophages, fibroblasts, and endothelial cells were incubated at 37°C in a humidified atmosphere chamber with 5% CO 2 , 21% O 2 , and 74% N 2 . The supernatant was discarded, non-adherent cells were removed, and specific fresh culture medium was changed every 2 days.
Adherent cells reaching 80% confluence were lifted with trypsin-EDTA solution (Gibco, Rockville, MD, USA) and then used in further experiments.

| Neutrophil migration assay
Fresh lung and adipose tissue neutrophils from obese animals, obtained as described above, were either stimulated with (1)  To determine the percentage of neutrophils that had migrated to the bottom chamber, a light microscope (Olympus) and a Neubauer chamber were used to count the number of cells that were able to migrate from the upper toward the bottom well, compared with the amount of cells that were seeded. Data represent the percentage variation related to regular medium (0.9% sodium chloride) from three independent experiments for each treatment condition. In alveolar macrophages, M2 markers, such as IL-10, TGFβ, and arginase, did not differ between the groups (Figure 2A   were not affected by any of the tested agents. Cells from lean animals did not show any alterations after exposure to these agents for 1 h. In short, propofol composition affected macrophages and neutrophils in lung and adipose tissue in obesity but not in lean animals.

| Statistical analysis
A diet-induced obesity model, previously characterized by our group, 13 presented increased body and trunk fat percentages, higher triglyceride, total cholesterol, and insulin and leptin levels compared with lean animals, as well as greater adipose tissue compartments normalized by body weight. This model was chosen since it resembles important aspects of human obesity, such as metabolic and hormonal changes. 13 In addition, this obese model can elicit low-grade inflammation as observed by increased levels of IL-6 in lung and adipose tissues, 13 suggesting that increased leptin levels may contribute to the release of pro-inflammatory mediators through macrophages. 20 Moreover, during obesity, immune cells may alter their receptor availability. 21 Propofol, a widely used anesthetic agent, is In cells from lean animals, no significant changes were observed between the groups, thus suggesting that the immune inflammatory response of obesity is necessary for the effects of active propofol and the lipid excipient.
This is the first study to evaluate propofol and its main formulation components separately considering the immunomodulatory properties in the scenario of low-grade inflammation in obesity. Our results have shown diverse effects among the immune cells tested and their origin, which may represent different functions, but they show a trend toward an anti-inflammatory profile on adipose tissue macrophages and neutrophils for the active components separately.
As lipid emulsions are also known to present immunomodulatory effects, 39

| Limitations
This study has some limitations: first, there is an important difference between human and murine neutrophils concerning chemokine receptor expression on the surface of neutrophils. Rodents do not express CXCL8, the major CXCR2 neutrophil ligand in humans.
Second, the dose was chosen according to the clinical application, 50 μM propofol. 10 Third, lipid excipients obtained from Cristália Laboratories may not have the same lipid components as the propofol purchased from another company. Therefore, further studies are required to evaluate the immunomodulatory effects of different propofol formulations, mainly in obese patients.

| CON CLUS ION
In the present in vitro study, in the obesity scenario, the active propofol component (2,6-DIPPH) and the lipid excipient have different immunomodulatory effects, which results in diverse effects of the overall formulation on the cells tested. The active propofol component presented diverse immunomodulatory effects depending on the immune cell, its origin, and context.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.

AUTH O R S' CO NTR I B UTI O N
LH, FC, PP, PS, and PR contributed to study conceptualization, data analysis, interpretation, and manuscript preparation. LH, FC, MA, CB, LA, RL, and SA contributed to experimental conduct, methods, and data analysis. LH, FC, MA, and SC contributed to mRNA data analysis.
LH, FC, PS, PP, and PR contributed to manuscript revision/finalization.
All authors had access to the data and reviewed the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets presented in this study can be found in online repositories. The details on the repository/repositories and accession number(s) can be found in the Supplementary Information.