Regulation of the Immune System: Hormones and Nerves 

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The Hypothalamic-Pituitary Adrenal Axis Overview

The Hypothalamic-Pituitary Adrenal (HPA) Axis is the name of the functional relationship between the hypothalamus, the key regulatory organ of the endocrine system, and the adrenal glands – hormone-producing tissue found bilaterally above the kidneys. One outcome of this system is the controlled production and release of glucocorticoids: a class of hormones that when activating their receptor, glucocorticoid receptor (GR), induce a myriad of effects in the body [74]. The HPA axis plays a major role in multiple systems: the cardiovascular system; metabolism; the nervous system and during neonatal development for lung maturation. Of particular interest to this investigation is the effect that the HPA axis has on the immune system and will be addressed in section 2.B.iii. Bioactive Endogenous Glucocorticoids – corticosterone in mice and cortisol in humans – acutely attenuate the immune response and versions of glucocorticoids have been used to treat inflammatory conditions for 70 years [74]. Considering the potent effect of glucocorticoids, it is no surprise that externally manufactured and exogenous glucocorticoids – prednisolone, dexamethasone and more – are widely used in modern medicine today [118]. This will be discussed at length in section 2.B.iv. The HPA-axis is a key regulator of immune function and will be discussed in full in the following sub-sections.

The HPA axis: Pathway

As stated in the previous section, the HPA axis consists of the hypothalamus signalling to the adrenal glands to produce and/or release glucocorticoid. In reality, the process is more complicated with multiple other factors playing a crucial role in the HPA axis. To summarise: the hypothalamus releases cortisol releasing hormone (CRH), which acts on the anterior pituitary to release adrenocorticotrophic hormone (ACTH) and then goes through the vasculature to the adrenal glands and releases glucocorticoid [Illustration.7]. This pathway will be discussed in detail here. The hypothalamus is a crucial component of the neuroendocrine system located at the base of the CNS and is only partially protected by the blood-brain barrier (BBB) [17], which is used to protect the CNS from peripheral stimuli. The overall architecture of the hypothalamus is the central out-growth of the CNS with a connecting stalk to the bulb of the pituitary gland – anterior and posterior. The anterior and posterior pituitary glands are important neuroendocrine glands with a variety of roles and responsibilities for hormone production throughout the body. Within the hypothalamus, there is a topographically discrete region called the paraventricular nucleus (PVN) [154]. The PVN consists of neurosecretory regions – the magnocellular and parvocellular PVN. Both the magnocellular and parvocellular PVN have primary roles, however there is some overlap between their functions [154]. The magnocellular PVN primarily produces vasopressin, a hormone that can induce constriction in arterioles of the vasculature and encourages water reabsorption in the kidneys. Whereas, the parvocellular PVN is responsible for producing and releasing CRH. CRH is released at the end of the parvocellular PVN neurons into the hypophyseal portal vein system at the base of the median eminence. This portal vein system allows for release of CRH into capillaries and then direct transfer, via portal vessels, into the capillaries of the anterior pituitary [154]. Within the anterior pituitary, CRH will bind to its receptor on the surface of corticotropes, which will induce the synthesis and release of Pro-opiomelanocortin (POMC) – the precursor of ACTH – and release of ACTH [126]. The adrenal glands are located bilaterally above the kidneys and can be divided into adrenal medulla and the adrenal cortex. The adrenal medulla is a modified tissue of the SNS and can produce the typical neurotransmitters of the SNS – epinephrine and norepinephrine, which are released into the blood and not, strictly, used a neurotransmitter. Whereas, the adrenal cortex is glandular tissue and is divided into three functionally distinct zones: zona glomerulosa to produce mineralocorticoids; zona reticularis to produce sex hormones and zona fasciculata to synthesise glucocorticoids. ACTH will bind to its receptor, mineralocorticoid receptor type 2, in the cells of the zona fasciculata, which induces downstream signalling, leading to the activation of cAMP-dependent protein kinase A (PKA). PKA, once activated, can regulate free cholesterol to aid in the production of steroid hormones and ultimately aid in the release of glucocorticoids [52].

The HPA axis: Instigating stimulus for the hypothalamus

The hypothalamus is in a somewhat unique position in the CNS, in that its activity can be regulated by the CNS, via nerves, or the periphery, via hormones and cytokines. The HPA axis, hypothalamic activity and ultimate glucocorticoid release, is regulated in a circadian manner. In humans, with a regular day-night routine, there is low glucocorticoid concentration in the blood by late evening and it rebounds after midnight to increase until peaking early morning [118]. The opposite is true in mice however, with their lowest levels during our morning. There is a region of the hypothalamus called the suprachiasmatic nucleus (SCN), which is responsible for the diurnal release of glucocorticoids. The SCN can track time autonomously but in order to function optimally it receives direct retinal innervation [62]. Additionally, the brainstem can send inputs to the hypothalamus to regulate activity [154]. These are the primary means by which the CNS regulates the hypothalamus, peripheral stimuli influencing the hypothalamus will now be discussed. In the literature it has been well described that a variety of peripheral stimuli can affect the activity of the hypothalamus [154]. The focus will be the specific regulation of CRH and ACTH in response to cytokines. Moreover, as multiple cytokine receptors and cytokines can have an influencing effect on CRH/ACTH secretion, the focus will be on IL-6, TNF and IL-1β. Within the hypothalamus there is expression of the mRNA of IL-6 and IL-6R [51] and there are binding sites for IL-6 in the anterior pituitary [116]. Furthermore, IL-6 expression is enhanced in both the hypothalamus and the pituitary following stimulation by LPS [90]. It has been demonstrated that IL-6 will increase the release of ACTH in vivo in rats [97], this increase remaining evident up to 24 hours later with the concentration of glucocorticoid also enhanced compared to control stimuli [61]. Interestingly, however, it is not indicative that IL-6 will increase the mRNA of CRH or POMC [61]. This result, perhaps indicates that IL-6 acting on its receptor, does not induce the production of CRH or ACTH, but only affects the release of already stored ACTH. The receptor for TNF has been discovered in two cell lines representing the pituitary [80] and TNF expression is found in the hypothalamus and pituitary gland in vivo after LPS injection [90]. In rats it has been demonstrated that TNF will increase the concentration of both ACTH and glucocorticoid in the blood. Furthermore, TNF will increase the secretion of CRH from primary hypothalamus cells tested in vitro and the increase of ACTH and glucocorticoid is blocked by CRH anti-serum [8]. This result indicates that TNF is acting primarily on the parvocellular PVN cells to release CRH, rather than directly on the pituitary. IL-1R was discovered in the murine pituitary gland [119] and LPS will increase the presence of IL-1β in the hypothalamus and pituitary [90]. Moreover, IL-1β can directly increase the mRNA of CRH and ACTH, as well as the secretion of ACTH and glucocorticoid even up to 24 hours after initial IL-1β injection into rats [61]. The relationship between IL-1β and the hypothalamus is particularly interesting, because IL-1β is one of the primary cytokines responsible for fever induction and the hypothalamus regulates temperature in the body. Researchers have investigated this relationship from multiple angles and this effect was crucial to the discovery of the inflammatory reflex, which will be discussed in section 2.C.ii. It is worth stating that there is a wealth of literature on cytokines affecting HPA activity and what is presented is only representative of the whole, while conflicting data can be found, it is well established that cytokines play a large role in the regulation of HPA activity [154].

Glucocorticoids in Medicine

Glucocorticoids have been in use since the late 1940’s in medicine: Today the use of glucocorticoids is one of the most widely prescribed drugs. Depending on the desired treatment, different forms of glucocorticoids can be used – hydrocortisone, prednisolone and dexamethasone. Hydrocortisone is the closest synthetic to human cortisol in activation potency and is considered low potency with 20 mg and with a half-life of 8-12 hours. Prednisolone is considered medium strength as its potency is 4 times greater than that of hydrocortisone at a 5 mg dose and a half-life of 12-36 hours. Whereas, dexamethasone is considered high potency as its anti-inflammatory activity is 30 times stronger than that of hydrocortisone at a dose of 0.75 mg and has a long half-life of 36-72 hours [118]. These are just examples of the many forms of synthetic glucocorticoids which can be for short- or long-term use. Additionally, glucocorticoids can be administered orally, topically, inhaled or injected intra-articularly. While, synthetic glucocorticoids are best known as medication against the immune response, there are some strictly non-immune related disorders treated by glucocorticoids. Sufferers of Addison’s disease generally require continuous administration of synthetic glucocorticoids such as hydrocortisone. Ambulatory surgery, or day surgery, is frequently complicated by post-operative and post-discharge nausea and vomiting, for which orally administered dexamethasone can give a sense of well-being, stimulate appetite and  reduce the occurrence of nausea or vomiting [147]. Routinely, glucocorticoids are used for their anti-inflammatory properties after surgeries and particularly for solid-organ transplants [96]. Furthermore, glucocorticoids are used for a large range of inflammatory disorders – sudden severe auto-immune disorder, respiratory infections and disorders, inflammatory skin conditions, but also IMID’s such as non-infectious colitis and arthritis [150]. The benefit of glucocorticoids is undoubtable, however there are risks associated with long-term use. There is considerable evidence for adverse effects due to long-term high-dose use of glucocorticoids [118], but even low-dose medium-term (>60 days) usage of glucocorticoids can cause adverse effects. In a study based in the United States with 2 446 active participants, 90% of the participants reported an adverse effect, with 55% stating it was bothersome. The predominant adverse effect was weight gain – 70% of all participants reported this outcome, but skin bruising and acne were also commonly reported. More serious conditions such as cataracts and fractures were also reported in a minority of the participants [35]. Long-term, high dose glucocorticoid use is a risk-factor for osteoporosis, T2D, hypertension, infection and iatrogenic Cushing’s syndrome [118]. Iatrogenic Cushing’s syndrome is an artificial or induced form of Cushing’s syndrome due to high-dose glucocorticoid treatment, the symptoms of which are like that of the physiological Cushing’s syndrome, but without the underlying pathology. Alternatively, there is also a risk of the opposite – iatrogenic adrenal insufficiency. Within the hypothalamus, as discussed, there is GR which glucocorticoids can bind to with the same efficiency as their target area. This activation in the hypothalamus performs the role of activating the negative feedback loop and prevents the production of CRH and ACTH, ultimately not producing natural glucocorticoids. This is a concern for patients who are going off long-term high dose usage of synthetic glucocorticoids, as it is possible a reset in the basal levels of cortisol has occurred [118]. Glucocorticoid administration can additionally be a risk factor for the immune system [59], even generating apoptosis in nearly all cells of the immune system in certain conditions [59, 173]. Overall the HPA axis and glucocorticoids are invaluable suppressors of the immune response, both physiologically and pharmacologically.

The Inflammatory Reflex: Vagus

The vagus nerve is the tenth of twelve cranial nerves, a group of ANS nerves that arise directly from the brainstem. It has an important role in multiple systems aside from the inflammatory reflex. Anatomically, the vagus can be found bilaterally running parallel to the trachea at the cervical level of intervention and additionally, sub-diaphragmatically, it runs parallel to the oesophagus. Furthermore, the vagus nerve splits and separates into various smaller nerves and innervates most major organs – the heart, lungs, liver, stomach, intestines, pancreas [11], non-organ ganglions – like the coeliac ganglion [125] and potentially, via the coeliac ganglion, the spleen [3]. Continuous vagal efferent stimulation inhibits the beat rate of the heart, which physiologically runs around 100 beats per minute. The efferent parasympathetic tone from the vagus keeps the heart rate closer to 60 beats per minute and this is perhaps the best characterised activity of vagal efferent fibres. It should be noted however that up to 90% of fibres within the vagus are afferent [11]. As widely dispersed as the vagus is throughout the body, at most nerve endings there is a sensor for the local environment, which will detect stimuli and induce afferent activity. In the lungs, there are neuroepithelial bodies which can function as mechanosensors for the CNS, which signal through vagal afferent fibres [91]. These mechanosensors can inform the CNS on breath rate and potentially induce an adjustment in the response. Additionally, there is a specific form of mechanoreceptors – baroreceptors, for measuring blood pressure – located in the aortic arch, which signal via afferent vagal fibres. Furthermore, there is also some evidence of chemoreceptors located in the rat aortic arch, though there is no evidence of what they are specifically detecting [26]. These diffuse vagal afferents re-combine with the core vagus nerves sub-diaphragmatically or at the cervical level, before the vagus arrives at the brainstem of the CNS and more specifically the nucleus tractus solarias (NTS) [47]. Interestingly, at the terminal of vagal afferents there are occasionally small tissues know as paraganglia. Paraganglia are non-neuronal cells derived from neural tissue during development. Vagal paraganglia are located densely at the cervical level, near the liver and comparatively sparsely near the pancreas. Paraganglia act as sensors of their environment and induce activity in the vagal afferent fibres [11]. Their specific role in regard to the inflammatory reflex will be outlined in the following section.

The Inflammatory Reflex – Afferent sensors

In this section, afferent vagal paraganglia will be discussed from a historical perspective. During the mid-1990’s, researchers were particularly interested in how IL-1β could induce the fever response – there were two hypotheses. The first was that IL-1β travelled to the CNS and acted on receptors there – particularly in the hypothalamus [12], in a similar manner to as outlined in section 2.B.ii. The second hypothesis was that IL-1β was being detected in the periphery by vagal nerves. In one study, it was shown that IL-1β will induce the fever response. The fever response, however, could be abolished by previous sub-diaphragmatic vagotomy (bilateral removal of the vagus nerve) in rats. More specifically, this result was replicable with hepatic branch vagotomy [165] – an area rich with vagal paraganglia. Therefore, these results indicate that IL-1β is acting on peripheral nerves to induce fever. This study launched the idea of peripheral nerves communicating inflammation to the brain. Continuing their research, the group next assessed if IL-1β administrated to the hepatic portal vein, a major blood vessel in the liver, induced activity in the afferent hepatic branch of the vagus. It was discovered, in a dose-dependent manner, that IL-1β will generate increased impulses per second in the hepatic branch nerve up to 60 minutes after initial injection [113]. This study provides strong evidence that the immune system can activate peripheral nerves, however it was still uncertain how this was happening. In a follow up study, the group assessed cryostat sections of the vagus nerve – whole, abdominal and liver. A staining was performed for IL-1R and it was discovered in multiple sections of the vagus nerve, particularly clustered in vagal paraganglia. This discovery lent further credence to their hypothesis that peripheral nerves sense IL-1β [55].

The Inflammatory Reflex – Efferent Response

Having outlined the hypotheses behind afferent activation of the inflammatory reflex in section 2.C.ii, the efferent response will be expounded upon, again using a historical perspective. The elucidation of the cholinergic anti-inflammatory reflex is in a large part thanks to Dr. Kevin Tracey and his team who have been involved in nearly all major discoveries for the last 20 years. Originally, Dr. Tracey was searching for a cure for septic shock and that is a focus in his early discussions. Since, however, the inflammatory reflex has expanded past the idea of septic shock. The original paper demonstrated that, in rats, isolation and electrical activation of the vagus nerve would attenuate LPS-mediated production of serum TNF, liver homogenate TNF and prevented the endotoxin-mediated decrease in mean arterial blood pressure – indicative of shock. Additionally, the authors likened the decrease of pro-inflammatory cytokines by ACh to ACh produced by the parasympathetic vagus nerve [152]. Overall this was an interesting study that launched additional research on the cholinergic anti-inflammatory reflex. The follow up paper from Dr. Tracey’s lab provided additional information on the cholinergic anti-inflammatory reflex: it established that electrical stimulation of the vagus nerve would mitigate LPS-induced TNF production in the heart, but not the lungs. Additionally, differences between left and right vagus were examined, interestingly left vagal stimulation (Left side – when animal is on stomach) was more effective than right vagal stimulation in managing mean arterial blood pressure in response to endotoxin challenge [9]. Currently, when the vagus is stimulated, the left nerve is typically used. In their next experiment, mice were used to test the inflammatory reflex. Wildtype mice receiving vagal electrostimulation had attenuated concentration of serum TNF, however α7nAChR (a sub-unit of the nAChR) knockout mice were incapable of replicating this response. In the study the result is likened to the fact that macrophages not expressing α7nAChR are incapable of responding to ACh and do not have decreased inflammation compared to control macrophages [162]. It was suggested, but not confirmed, that macrophages expressing α7nAChR mediate this effect. Overall, this study adds to the developing mechanism of action associated with the cholinergic anti-inflammatory reflex. A study by a separate group, in the same year, demonstrated the importance of the nAChR to the cholinergic anti-inflammatory reflex using agonists. Chlorisondamine, an antagonist of nAChR, reversed the decrease of liver TNF mRNA, serum TNF and NF-κB expression by rat vagus electrostimulation [60]. While it is known now that the spleen is a crucial component of the inflammatory reflex, it was not known in 2003, when an independent group were investigating splenic nerve stimulation. An ex vivo perfusion preparation of the spleen was performed: The spleen was kept intact, as well as the surrounding tissue and particularly the splenic nerve – which was electrostimulated. It was shown that LPS-mediated production of TNF was attenuated due to the electrostimulation and it was wholly mediated by βAR [77]. This result indicates that there is sympathetic regulation of inflammation via nerves, in addition to parasympathetic. Interestingly, this paper gives a hint as to what the cholinergic anti-inflammatory reflex would become, but it also is the basis of an alternative theory on the inflammatory reflex, which will be discussed in the following section 2.C.iv.

Table of contents :

Description Page Number
French Abstract 2-3 English Abstract
Acknowledgments
Table of Contents
Abbreviation list
Guide to Introduction
Introduction
1.0.0 – Immune System 
1.A.0 – Overview
1.B.0 – Innate Immune Response
1.B.i – Cells
1.B.ii – Cytokines
1.C.0 – Adaptive Immune Response
1.D.0 – Dysregulated immune system
1.E.0 – Summary
2.0.0 – Regulation of the Immune System: Hormones and Nerves 
2.A.0 – Hormones and nerves introduction
2.B.0 – HPA – Overview
2.B.i – Pathway
2.B.ii – Instigating stimulus for hypothalamus
2.B.iii – Glucocorticoids
2.B.iv – Glucocorticoids in Medicine
2.C.0 – The Inflammatory Reflex
2.C.i – Vagus Nerve
2.C.ii – Afferent Sensors
2.C.iii – Efferent Activity
2.C.iv – Evolving theory
2.D.0 – Summary
3.0.0 – Carotid Body 
3.A.0 – Overview
3.B.0 – Anatomy
3.B.i – Molecular and cellular
3.C.0 – Physiological functions
3.C.i – O2 sensing
3.C.ii – Metabolism
3.C.iii – Blood pressure
3.C.iv – Immune system
3.D.0 – Summary
4.0.0 – Bioelectronic Medicine 
4.A.0 – Overview
4.B.0 – Bioelectronic medicine and the vagus
4.B.i – Pre-clinical: Inflammation
4.B.ii – Clinical: Inflammation
4.C.0 – Bioelectronic medicine and the Carotid Body
4.C.i – Pre-clinical: Metabolism
4.C.ii – Clinical: Blood pressure
4.D.0 – Future of Bioelectronic Medicine.
Hypothesis & Aims

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