Endotoxin-induced Inflammatory and Behavioral Responses and Predictors of Individual Differences

Endotoxin-induced Inflammatory and Behavioral Responses and Predictors of Individual Differences: A Randomized, Double-blind Placebo Controlled Study on Healthy Human Volunteers

The objective of the present study is to specifically assess the effect of lipopolysaccharide (LPS) administration on the development of behavioral symptoms and the underlying contribution of inflammatory processes. In particular, the investigators will assess the development of subjective and objective behavioral symptoms. In addition, the investigators will determine whether some psychological trait or state can predict and/or modulate the LPS-induced inflammatory and behavioral responses. Twenty-five healthy subjects will be included. A placebo-controlled, double-blinded and cross-over design will be used. Subjects will receive an intravenous injection of endotoxin at 2 nanogram/kilogram (ng/kg) of body weight and an intravenous injection of sodium chloride as placebo of endotoxin injection at two different occasions. Prior to inclusion and randomization, subjects will come at the hospital and will receive a medical examination. Psychological variables that could affect the behavioral (or immune) response to LPS will be assessed at that time, using several self-assessment questionnaires. On the trial days, injection of endotoxin or sodium chloride will be performed and blood samples will be taken just before the endotoxin or sodium chloride injection and 1, 1.5, 2, 3, 4, 5, 6 and 7.5 hours after the injection. Blood samples will be used to measure several inflammatory and immune markers. Urine samples will be taken before the endotoxin or sodium chloride injection and as late as possible after the injection. Subjects will wear T-shirt all day. Urine and T-shirt samples will be used for behavioral assessment and analysis of body odor compound. Self-assessment questionnaires assessing behavioral and psychological variables will be completed by participants just before the endotoxin or sodium chloride injection, three hours and 7.5 hours after the injection. A short questionnaire assessing sickness behavior (SicknessQ) will be repeatedly completed by participants from just before to 7.5 hours after the endotoxin or sodium chloride injection. Several behavioral tests will be used, including a motivation task, a test assessing behavioral response to negative and sickness stimuli. Analysis of gait and motion, as well as of social interactions, will be performed. Photographs will be taken for the further rating of the faces.

Study design Twenty-five subjects will be included in the present study, which will follow a prospective, placebo-controlled and cross-over design. Participants will be blinded, as well as research personnel in charge of the participants and the laboratory. Subjects will be randomly assigned in the cross-over design with two trials, with about four weeks wash-out between LPS stimulations: Trial 1: endotoxin. Subjects will receive an intravenous injection of endotoxin at 2 ng/kg of body weight. Trial 2: placebo. Subjects will receive an intravenous injection of sodium chloride as placebo of endotoxin injection. Ten other healthy volunteers will be recruited in a pilot study prior the beginning of the main study, in order to test the behavioral tasks that will be used. These subjects will be recruited among the university staff or among university students. They will perform four specific behavioral tasks and will complete the questionnaires assessing psychological and behavioral variables in order to associate outcomes from the behavioral tasks to psychological and subjective behavioral assessments. No biological samples (blood, urine or tee-shirt samples) will be taken in subjects recruited in the pilot study. Treatment The LPS is provided by LGC Promochem AB, Albanoliden 5, 4tr, 506 30 Borås, Sweden. The USP (United States Pharmacopeial Convention) Endotoxin (Cat. No. 1235503) does come as a lyophilized (freeze-dried) powder and each vial contains 10,000 USP Endotoxin Units. Each vial contains very approximately 1 microgram of Endotoxin and is mixed with 5 milliliter (mL) of sterile water. Study procedure The study will be conducted at Department of Clinical Sciences, Danderyd Hospital. Prior to inclusion and randomization, subjects will come at the hospital in order to be subjected to a medical examination, including an electrocardiogram and blood laboratory analyses. Socio-demographic data and medical history will be assessed at this occasion, as well as body variables (weight, height, body composition) and physical activity using the Swedish version of the IPAQ (International Physical Activity Questionnaire). History of major depression will be assessed using the Mini-International Neuropsychiatric Interview (M.I.N.I.) for major depression. Psychological variables that could affect the behavioral (or immune) response to LPS, and which are not believed to change between the screening day and the trial days, will be also assessed at that time, using several web-based self-assessment questionnaires. These measures will include the subjects' perception of the body and health risk (e.g., interoceptive awareness, perceived vulnerability to disease, health anxiety and disgust reactivity, somatosensory amplification, fear of anxiety-related sensations) and psychological well-being (e.g., perceived stress, optimism, social support, cognitive well-being). The overall time for the medical examination will be of 30-45 minutes. The time to complete questionnaires is estimated to approximately 45 minutes but can be done from home. The participants will also receive a kit with a T-shirt (with sewn-in cotton pads) and a scent-free soap. Participants will be asked to wear the T-shirt in the morning (after washing armpits) on each trial day, and take it off after their arrival. This will allow collecting pre-treatment (baseline) samples of body odor. Participants will then be subjected to the two trials performed in a randomized order, with at least six weeks of wash-out: endotoxin versus placebo. The participants will be asked to restrain from strenuous exercise and alcohol taking 48 hours before the trial day, to avoid strong spices 24 hours before the trial day, and to take a light breakfast the morning of the trial day. Participants will arrive on the morning (at 7.30AM). They will turn in their morning T-shirt (see above) and change to a new T-shirt (with sewn-in cotton pads in the armpit) to wear during all day. The T-shirts will be used for behavioral assessment and analysis of body odor compound. Shortly after their arrival, two intravenous catheters will be placed: one for blood sampling at one arm and one at one hand for the injection of endotoxin or sodium chloride that will be removed after the injection. An anesthetic cream will be provided to spread on their arm in order to minimize the pain of venipuncture. Subjects will then rest for at least 30 minutes. Injection of endotoxin or sodium chloride will be performed when the rest is completed (around 8.30AM). Blood samples (10 mL in EDTA (Ethylenediaminetetraacetic acid) tubes and 10 mL in sodium citrate tubes) will be taken just before the endotoxin or sodium chloride injection and 1, 1.5, 2, 3, 4, 5, 6 and 7.5 hours after the injection. In addition, 2.5 mL in PAXGene™ tubes will be taken before and 2, 4 and 7.5 hours after the injection. Two additional samples in EDTA tubes (8 mL of blood) will be taken before the injection and 3 and 7.5 hours after the injection. The total quantity of blood taken will be of about 200 mL, corresponding to a third of the amount given at a blood donation. Blood samples will be used to measure concentrations of pro- and anti-inflammatory cytokines and other inflammatory markers (e.g., C-Reactive protein - CRP) using Enzyme-Linked Immunosorbent Assay (ELISA), immune cell subpopulations and concentrations of microparticles using flow cytometry, genetic markers using Cap analysis gene expression (CAGE) analyses and white blood cell count and differential. Urine samples will be taken before the endotoxin or sodium chloride injection (i.e., before the catheters are placed) and as late as possible after the injection (classically around four hours after the injection). Participants will be monitored with continuous electrocardiogram. Oxygen saturation (SO2), blood pressure and body temperature will be monitored every 30 minutes. Nitrogen dioxide (NO2) will be monitored continuously, methemoglobin checked at the same time than each blood sample. Self-assessment questionnaires assessing behavioral and psychological variables will be completed by participants just before the endotoxin or sodium chloride injection (during the rest period), three hours and 7.5 hours after the injection. A short questionnaire assessing sickness behavior (SicknessQ) will be repeatedly completed by participants from just before to 7.5 hours after the endotoxin or sodium chloride injection. Subjects will be filmed when in bed and will wear a sociometric badge, in order to analyze participants' behavioral response to a standardized situation of social interaction. Photographs will be taken before (i.e., before the catheters are placed), two hours after and 7.5 hours after the injection, for the further rating of the faces (e.g., health, attractiveness, tiredness). In parallel, skin color will be measured at the same time than blood samples (except T1.5) in order to assess a potential mediating effect of faces' health ratings. Analysis of subjects' gait and motion will be performed two and half hours after the injection. Short computerized cognitive tests will be performed after these tests, just before three hours after the injection. Subjects will perform a motivation task after completing the questionnaires three hours after the injection. In addition, two tests assessing behavior in response to positive/negative and healthy/sickness stimuli (an approach-avoidance behavior test and a regulation of emotion test) will be executed three hours and half after the injection. Participants will be allowed to take a light meal (snack and juice drink) around lunch time (12.30) and will hand in their T-shirt just before five and half hours after the injection. The last blood samples will be taken 7.5 hours after the injection and participants will be discharged. Monitoring of the experiment will be done by a medical doctor, supervised by a specialist in anesthesia and intensive care.

Change in symptoms of anxiety evaluated using the self-assessment questionnaire State part of the State-Trait Anxiety Inventory (STAI)

Change in mood alterations evaluated using the self-assessment questionnaire Swedish Core Affect Scales (SCAS)

Change in symptoms of pain evaluated using the self-assessment questionnaire Mc Gill questionnaire - short version

Change in plasma concentration of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1ra)

Change in headache scores measured using a numerical scale ranging from 0 (no headache) to 10 (most unbearable headache)

Change in nausea scores measured using a numerical scale ranging from 0 (no nausea) to 10 (most unbearable nausea)

Change in back pain scores measured using a numerical scale ranging from 0 (no back pain) to 10 (most unbearable back pain)

Changes in concentrations of total microparticles (defined as particles less than 1μm in size and staining for lactadherin) and platelet-, monocytes- and endothelial-derived microparticles (co-staining of lactadherin and, respectively, cluster of differentiation (CD)42A, CD14 and CD62E).

Change in expression of immune cell subpopulations markers using flow cytometry from frozen whole blood in 10% Dimethylsulfoxid (DMSO)

Change in abundance of volatile odor compounds analyzed using gas chromatography mass spectrometry (GC-MS) from tee-shirt samples and urine samples

Inclusion Criteria: Healthy subjects Exclusion Criteria: Diagnosed physiological or psychiatric disease Needle anxiety or blood phobia Regular medication (excluding contraceptive pill) Infection in the last two weeks Pregnancy or breastfeeding Smoking Excessive alcohol use Body mass index in the range of obesity (>30 kg/m2) or underweight (<18.5 kg/m2) Invisible veins in the antecubital area of the arms

Lasselin J, Petrovic P, Olsson MJ, Paues Goranson S, Lekander M, Jensen KB, Axelsson J. Sickness behavior is not all about the immune response: Possible roles of expectations and prediction errors in the worry of being sick. Brain Behav Immun. 2018 Nov;74:213-221. doi: 10.1016/j.bbi.2018.09.008. Epub 2018 Sep 11.

Axelsson J, Sundelin T, Olsson MJ, Sorjonen K, Axelsson C, Lasselin J, Lekander M. Identification of acutely sick people and facial cues of sickness. Proc Biol Sci. 2018 Jan 10;285(1870):20172430. doi: 10.1098/rspb.2017.2430.