Effects of Cold Exposure and Breathing Techniques on Immune Response
Condition: Cold Exposure; Hyperventilation; Inflammation; Endotoxemia
MeSH terms: Endotoxemia; Hyperventilation; Inflammation
Study type: Interventional

Summary: 
      Inflammatory cytokines play a pivotal role in rheumatoid arthritis (RA) and innovative
      non-pharmacological therapies aimed at limiting cytokine production are highly warranted.
      Recently, our group showed that healthy volunteers trained in an intervention developed by
      'Iceman' Wim Hof were able to voluntarily attenuate the pro-inflammatory response during
      experimental human endotoxemia (a model of systemic inflammation elicited by administration
      of lipopolysaccharide [LPS] in healthy volunteers). Subjects trained in the intervention
      exhibited profound increases in plasma adrenaline levels, a rapid increase of an
      anti-inflammatory cytokine and subsequent attenuation of the pro-inflammatory response.

      The intervention consists of three elements, namely meditation, exposure to cold and
      breathing techniques. The meditation element is not likely to be involved. It was a very
      minor part of the training program and was not practiced during the endotoxemia experiments.
      Exposure to cold and the subsequent rewarming to normal body temperature may influence the
      inflammatory response through the release of immunomodulatory molecules like HSP-70. Also,
      exposure to cold can induce an ischemia-reperfusion-like state in the skin and peripheral
      tissue that is known to be involved in the downregulation of pro-inflammatory cytokines and
      upregulation of anti-inflammatory cytokines. The investigators anticipate that the third
      element, breathing techniques, is the major contributor to the anti-inflammatory effects of
      the intervention previously observed. The present study aims to explore the effects of the
      breathing technique ('strength ventilation'), the exposure to cold, and these two elements
      combined on the immune response during human endotoxemia. Elucidation of the relative
      contribution of the elements is of importance to establish a feasible, safe, and effective
      intervention for future use in patients.

      Objective: The primary objective of the present study is to determine the effects of the
      'strength ventilation' breathing technique and exposure to cold, both separately and in
      combination, on the inflammatory response during human endotoxemia. To this end, a 2 by 2
      design will be employed. Additionally, an evaluation of the influence of the cold exposure
      and breathing technique on pain thresholds and oxygen tension in the mitochondria will take
      place.
    

Detailed description: 
      Auto-immune diseases are characterized by an inappropriate inflammatory response against
      tissues in the body. These diseases, of which rheumatoid arthritis (RA) is the most
      well-known, represent a major health care burden. Pro-inflammatory cytokines such as TNF-α,
      IL-6 and IL-1β are central in the pathogenesis of RA and many other auto-immune diseases.
      Biologics that antagonize inflammatory cytokines or their receptors, e.g. anti-TNF-α, soluble
      TNF-α-receptor, anti-IL-6 receptor, and IL-1 receptor antagonist, are very effective
      treatments. However, they are very expensive and can have serious side effects. Furthermore,
      the consequences of RA are currently completely beyond the patients' sphere of influence.
      Therefore, innovative therapies aimed at limiting inflammation in RA patients are warranted.

      Recently, a study into the effects of an intervention developed by 'The Iceman' Wim Hof
      revealed that it is possible to voluntarily attenuate the pro-inflammatory response during
      experimental human endotoxemia (a standardized, controlled, and reproducible model of
      systemic inflammation elicited by administration of lipopolysaccharide (LPS) in healthy
      volunteers). The intervention developed by Hof consists of several elements, namely
      meditation, exposure to cold and breathing techniques. Subjects trained in this intervention
      exhibited profound increases in plasma adrenaline levels, a rapid increase of the
      anti-inflammatory cytokine IL-10, and subsequent attenuation of the pro-inflammatory response
      (e.g. plasma levels of TNF-α, IL-6, and IL-8) during experimental human endotoxemia. This
      intervention could therefore represent a treatment modality that would empower RA patients to
      exert self-control over their disease.

      Based on these data, investigating the effectiveness of the intervention in RA patients is
      highly warranted. The study described in this protocol is part of a larger project in which
      the investigators ultimately strive to translate the intervention to clinical practice for RA
      patients. However, there are important issues that need to be addressed first. For example,
      feasibility and safety would be substantially improved if patients would only have to learn
      or practice one of the three elements of the intervention. The meditation element is not
      likely to be involved. It was a very minor part of the training program and was not practiced
      during the endotoxemia experiments. Also, there is no objective manner to measure this
      element and there is no hypothesis for a possible mechanism. Concerning the second element,
      exposure to cold, it would especially be of value to determine whether this element has
      additional value, because it is very demanding and might not be suitable for RA patients at
      all. The investigators anticipate that the third element, breathing techniques, is the major
      contributor to the anti-inflammatory effects of the intervention previously observed for
      reasons outlined below.

      Breathing techniques First, although the healthy volunteers were trained in all three
      elements, subjects only practiced breathing techniques during the endotoxemia experiment.
      Second, the breathing techniques were characterized by cyclic hyperventilation, which has
      been shown before to result in increased adrenaline levels. Subjects practiced two types of
      breathing techniques during the endotoxemia experiment in our previous endotoxemia study.
      Both of these breathing techniques were characterized by cycles of hyperventilation. In one
      of the techniques (hyper/hypoventilation), subjects held their breath for up to several
      minutes after each hyperventilation period, while in the other technique (strength
      ventilation), subjects held their breath for only 10 seconds during which all muscles were
      tightened after each hyperventilation period.

      Yet unpublished data (CMO 2014-1374) showed the effects of these two breathing techniques in
      the absence of cold exposure (or meditation) on plasma adrenaline levels. The investigators
      found these to be strongly correlated with the anti-inflammatory effects previously found.
      Both breathing techniques resulted in comparable increases in plasma adrenaline levels.
      Furthermore, adrenaline levels in subjects trained by Hof were comparable to those that were
      trained by an independent trainer previously not familiar with the intervention developed by
      Hof. Finally, adrenaline levels in subjects trained for 4 days were similar to those who were
      trained for only 2 hours.

      Based on these results, it is hypothesized that the strength ventilation technique is the
      major contributor to the anti-inflammatory effects of the intervention previously observed.
      However, the effects of solely this breathing technique on the inflammatory response is not
      investigated yet. Furthermore, it cannot be ruled out that the exposure to cold has
      additional effects, since there are several ways in which exposure to cold could contribute
      to anti-inflammatory effects, as described below.

      Exposure to cold First, exposure to cold and the subsequent rewarming to normal body
      temperature may influence the inflammatory response through the release of immunomodulatory
      danger associated molecular patterns (DAMP's), more specifically TLR-4 ligands such as heat
      shock protein 70 (HSP-70). It was shown that HSP-70 mRNA levels in isolated cardiac myocytes
      increased during rewarming after 2.5 hours of hypothermia. Furthermore, pre-incubation of
      human cells exposed to 4 °C for 1, 2, 3 and 4 hours induced synthesis and accumulation of
      HSP-70 upon recovery to 37 °C. The relevance of HSP-70 for the inflammatory response is
      evident from a study in which HSP-70 was shown to induce potent anti-inflammatory effects
      resembling induction of endotoxin tolerance in human monocytes.

      Second, exposure to cold can induce an ischemia-reperfusion-like state in the skin and
      peripheral tissue. A study into the effects of cryotherapy showed that local cooling of the
      skin decreases local tissue perfusion. The combination of tissue hypoperfusion and
      reperfusion upon the reactive vasodilatation that follows after rewarming is a form of
      ischemia/reperfusion (I/R). The potency of I/R to protect tissue against ischemic damage is
      known as Ischemic Preconditioning (IPC). It has the potential to influence the immune
      response through several pathways. For instance, recent animal work has shown that I/R
      results in downregulation of pro-inflammatory cytokines such as TNF-α and IL-6 and
      upregulation of anti-inflammatory cytokines such as IL-10. Furthermore, hypoxia-inducible
      factor (HIF) has been shown to be a major contributor to the I/R-induced IL-10 response.

      Third, exposure to cold could have a potentiating effect on the adrenaline release evoked by
      the breathing techniques. In a study of human volunteers, adrenaline levels were increased
      after 2 hours of sitting in a cold room. Interestingly, in a study during acute exercise in
      human volunteers, cold exposure prior to exercise was associated with an added
      immuno-stimulatory effect. Also, cytokineresponse of IL-1beta and IL-6 in ex vivo
      LPS-stimulated blood was lower in experienced ice-swimmers compared to inexperienced
      ice-swimmers.

      Taken together, cold exposure may influence the in vivo response to endotoxemia. This might
      be mediated by direct, adrenaline-independent effects or by enhancing adrenaline levels
      elicited by strength ventilation.

      Synthesis This study aims to explore the effects of two elements of the intervention
      initially developed by Hof, namely the strength ventilation breathing technique and the
      exposure to cold, on the immune response during human endotoxemia. Effects of both elements
      separately as well as in combination will be tested, the latter to explore the interplay
      between the elements.

      In addition, an assesment will be made of the effects of the (combination of) breathing
      techniques and cold exposure on pain perception. In previous studies, trained subjects
      experienced substantially less flu-like symptoms during endotoxemia. This could be due to the
      attenuated immune response in these subjects, but also through other effects induced by the
      breathing techniques and/or cold exposure. To investigate this, quantitative sensory testing
      (QST) is used, an objective technique to measure pain thresholds.

      Finally, non invasive measurements of oxygen tension in the mitochondria will be used to
      asses mitochondrial function during human endotoxemia by using the Protoporphyrin IX-Triplet
      State Liftime Technique (PpIX-TSLT).

      Mitochondrial dysfunction is an important element in the pathophysiology of sepsis. However,
      a valid non-invasive method to measure mitochondrial function is not yet available. In animal
      models, the PpIX-TSLT technique has shown to be a feasible technique to measure alterations
      in mitochondrial oxygen tension during endotoxemia. This has never been studied in humans
      during endotoxemia. Furthermore, the exposure to cold and especially the breathing technique
      may influence oxygen tension in the mitochondria as well, as blood gas parameters (e.g. pCO2,
      acid base balance etc.) fluctuate to a large extent during the practicing of this technique.
    

Intervention name(s): Cold Exposure; Strength Ventilation
Intervention type(s): Behavioral; Behavioral
Intervention description(s): The start of the training is scheduled 7 to 12 days before the endotoxemia experiment day. Subjects in this group will participate in a 4 day intensive cold exposure course (see section 5.1). On day 1, venous blood will be sampled directly after ice water immersion. On day 4, venous blood will be sampled before the training procedures on that day and after the last ice water immersion.
After the 4 day course, subjects will continue to practice cold exposure at home using cold showers. The timing and length of this training is analogous to the time spend on training in the cold in our previous study protocol [7].; The training is scheduled 2 to 6 days before the endotoxemia experiment day. Subjects in this group will receive a detailed, written instruction about the strength ventilation technique (see section 5.1). In addition, they will receive a 2 hour instruction session during which the research team will supervise the practices and clarify the instructions of needed. Subjects are instructed not to practice the learned techniques at home.
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: A parallel randomized controlled explorative study in healthy male volunteers during experimental endotoxemia.
Masking: Single (Outcomes Assessor)
Primary purpose: Basic Science