Acute Exercise and NK Cell Regulation in Tissue and Circulation After IL-6R Blockade
Condition: Interleukin-6 Inhibition; Physical Stress; Appetitive Behavior
Study type: Interventional

Summary: 
      The study is a randomized controlled trial studying the acute effect of intense aerobic
      exercise upon NK (Natural Killer) cell activation and appetite-regulation in human
      participants with or without concommitant IL-6R receptor blockade (Tocilizumab)
    

Detailed description: 
      Objectives:

      Primary Objective:

        -  To explore the association between acute exercise, IL-6 blockade and NK regulation in
           circulation.

        -  To explore whether exercise activated NK-cells have a unique phenotype using single cell
           RNA sequencing.

      Secondary Objectives:

        -  To explore the effect of acute aerobic exercise on NK cell number and activity in
           circulation, muscle and adipose tissue, in healthy young males, in order to indirectly
           explore exercise as an anti-cancer remedy.

        -  To assess the kinetics of the NK-cell response and, if possible, chronologically
           investigate the appearance and whereabouts of the activated NK-cells.

      Explorative Objectives

        -  To establish a reproducible protocol for tracking various immune-cells and their
           involvement in the acute exercise response in humans

        -  To investigate novel signal molecules released from muscle during exercise with
           immunological importance.

        -  To investigate the possible role of exercise induced IL-6 on subjective feelings of
           hunger and satiety post exercise and food intake. • To explore the effect of IL-6
           receptor blockade on the exercise proteome and metabolome, using mass spectrometry

      Methods:

      30 healthy recreationally active young males will be included in this acute exercise study in
      which NK-cell kinetic and regulation will be studied in response to acute aerobic exercise
      and IL-6R blockade or placebo.

      The study consists of 2 visits. At inclusion (visit 1) all subjects will undergo assessment
      encompassing: baseline medical screening (auscultation, blood pressure, ECG), determination
      of body composition (DXA), cardiovascular fitness (VO2max) and standard fasting
      blood-biochemistry which will be analyzed immediately. Visit 2 will consist of a muscle and
      fat biopsy from the dominant leg and abdominal subcutaneous-fat depot respectively (both
      under resting conditions), which will then act as reference tissue for the further tissue
      analysis. Then, a 18G antecubital peripheral venous access will be secured prior to
      commencing with IL-6R infusion. 2 hours after the infusion procedure the subjects will
      undergo an acute exercise protocol. Subjects will then be challenged by a high intensity
      acute aerobic exercise bout, utilizing a bicycle ergometer. Blood samples will be drawn prior
      to the infusion and exercise, during exercise, as well as immediately, ½, 1, 1½ and 2h post
      exercise and up 4h post exercise in the appetite regulation substudy. Blood samples will
      immediately be analyzed for leucocyte count and differentiation as well as plasma
      biochemistry. In addition, blood samples will be utilized for NK cell isolation with
      subsequent single cell RNA sequencing, immune cell distribution and killing capacity towards
      cancer cells. Furthermore, plasma samples will be collected and frozen for later
      determination of cortisol, prolactin and circulating cytokines, including but not limited to
      IL-6 and G-CSF. Lastly, in the appetite substudy, insulin, GLP-1 and free fatty acids will be
      measured.

      Tissue samples will be obtained 2h post exercise for optimizing gene-expression analysis.
      Both muscle and adipose tissue samples will be analyzed for inflammatory and
      anti-inflammatory markers, NK cell content and phenotyping of these NK cells using markers
      obtained from single cell sequencing, conducted on the blood-borne NK-cells. Furthermore,
      immune cell infiltration will be assessed using histology. All tissue samples will be
      obtained using a Bergström needle under sterile conditions during local anesthesia in order
      to minimize any sample-related discomfort or infection.

      After the tissue sample, an ad libitum meal will be served, the subjects can eat as much can,
      but are instructed not to overconsume as any leftover food can be taken home. Paracetamol
      (1.5g) will be given to asses gastric emptying.

      All participants will undergo the 2 study visits at the same approximate time of the day
      (9.00 a.m.) After completion of the study, any left-over material will be transferred to the
      CFAS biobank.

      Subjects: Included subjects will be 30 recreationally active, moderately trained, healthy
      young males aged 18-40 years. Exclusion criteria are: cardiovascular, rheumatologic and
      metabolic disease, elite sports or high aerobic training status. Chronic use of nonsteroidal
      anti-inflammatory drug (NSAID) or other immunosuppressants.

      Intervention: Subjects will be randomized to acute exercise, with (n=15) or without (n=15)
      prior IL-6R blockade. The exercise intervention will consist of a highly taxing ≈45 minutes
      interval based, aerobic exercise bout, conducted on a stationary bicycle ergometer. After an
      initial 5-minute warmup at 50-60% of HRmax, subjects will undergo seven, verbally encouraged,
      3-minute intervals at above 90% HRmax, interspersed by 3 minutes of low intensity pedaling.
      Blood samples will be drawn before, during as well as immediately, ½, 1, 1½ and 2h post
      exercise together with tissue sampling at the 2h time-point.

      To assess appetite regulation, further blood samples will be drawn from 2h-4h post exercise.

      Statistical considerations: Based on data from our pilot study, we anticipate a ≈ 45 % lower
      maximal NK cell mobilization in the IL6R inhibition group compared to CON and a 29 %
      difference in NK cell nadir with the lowest values obtained in the IL6R inhibition group.
      Assuming a 5%-significance level in two-sided tests, we need to include 9 patients in each
      group to achieve 90% power for detecting a relative difference of 45 % in the
      intervention-group. To account for potential dropouts/excluded, due to the somewhat invasive
      setup, we will include a total of 30 patients (15 per group).

      Recruitment: Subjects will be recruited through forsøgsperson.dk or similar sites alongside
      advertising at relevant sites.

      Subjects will contact us at CFAS and will be given the choice whether to receive study
      information pr. telephone (thereby denying the option of a bystander) or a face to face
      meeting at CFAS. All this information will be given by the primary investigator. If the
      subject is interested in hearing more of the study, relevant documents will be emailed. If
      the subject is interested in joining the study after having received written or oral
      information, he will have 24h to consider study participation. When the subject has been
      informed either by telephone or face to face and accepts study participation, he will be
      invited to visit 1 where written and informed consent will be obtained (or signed and brought
      from home by the participant).

      Risks and adverse reactions: Subjects may experience minor discomfort with regard to blood
      and tissue samples. The blood volume drawn is negligible and will not be associated with any
      health risk. All sampling of tissues will be performed under sterile conditions during local
      anesthesia and will thus be associated with little pain or discomfort and very little risk of
      infection. During exercise testing, participants may experience shortness of breath.

      IL-6R inhibition is generally well tolerated with only few side-effects. The dose of
      paracetamol is low and is not associated with any risk.

      Contact information of the involved medical doctor will be given to participants at study
      inclusion, so that any adverse event can be reported and solved.

      Dissemination of study results: Both positive, negative and inconclusive results will be
      published in relevant international scientific journals.

      Ethical consideration: The project is expected to cause limited risks, side effects and
      discomfort. All procedures will be performed by experienced physicians and physiologists with
      relevant safety. Tocilizumab is generally well tolerated and the dose of paracetamol is low.
      Included subjects may at any time, and without justification, retract their consent of study
      participation. We believe that the project is important and will contribute with critical new
      information on the IL-6 modifiable NK-cell response to acute exercise both in circulation and
      in adipose and muscle tissue (as there is currently very limited knowledge on the latter).

      The study is considered a toolbox-study by Lægemiddelstyrelsen, and hence not at
      pharmaceutical study
    

Intervention name(s): Tocilizumab Injectable Product; Saline 0.9% 100 ml NaCl 0.9% will be infused over 1 hour
Intervention type(s): Drug; Other
Intervention description(s): The intervention group will undergo intense aerobic exercise with prior IL-6R infusion for 1h prior to the exercise bout.; The control group will undergo intense aerobic exercise with saline infusion for 1h prior to the exercise bout
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Triple (Participant, Investigator, Outcomes Assessor)
Primary purpose: Basic Science
Keywords: Natural Killer cells; Aerobic exercise; IL-6R blockade; Human; Appetite