BME 620 Feasibility Analysis by Sz-Wei Wu Topic: “An Electroceutial Method to Tackle Diseases by Controlling the Relationship Between Immune System and Nervous System ”

I. General Introduction Health is the most paramount asset for each person, not matter at physical or mental part. Without a healthy body, people generally cannot and will not have a nice quality of life. As modern human population has evolved around 200,000 years, we have gained multiple mechanisms to protect and maintain our health by innate and adaptive immune system. With the advance of modern medicine, we have more understanding of our physiological and anatomical system. We have discovered and/or invented utilizing vaccine for prophylactic, nutrient supplements for maintenance, medication to ameliorate or cure diseases. All of the methods need to work and tune closely with our delicate immune system; otherwise, it might lead to a catastrophic result. Another amazing system assists us monitoring our health automatically and maintaining our body in a homeostatic condition is the nervous system. Although we have attained a remarkable achievement with medication so far, there is always an intriguing question whether we can more directly manipulate these two systems as we want and parse the communication between them. The answer is yes, it is possible and feasible to utilize bioelectronics techniques to achieve the goal, supported by the emerging evidences from several research groups’ results in these two decades around the world, but there are still many issues need to conquer before the general people can embrace the fruit, such as technology, regulation and even morality. I believe that, in the near future, we will see the adoption with the promising and continuing advancement at the process of semiconductor industry, packaging and lead design for implantable device, energy harvesting techniques, algorithm to tune the optimized parameters and process the data, flexible mircro-electrode array design and understanding the mechanism of neuromodulation. In this report, I will introduce the current development of neuromodulation about immune applications focusing on vagus nerve stimulation (VNS) with the behind mechanism, analyze the current trend in the industry, and propose a device that can help us gain a more objective and comparable result. Before the detail contents, I list several statistical numbers from different reports to substantiate why we need to do more research on it and where is the potential market if you look at a profitable point of view. According to the statistics, the United States spent $2.9 trillion on healthcare alone i.e., $9,255 per person in 2013 [1]. A substantial portion of costs can be attributed to diseases related to immune response such as influenza, infection, asthma, allergy, rheumatoid arthritis, autoimmune disease, diabetes, sepsis, cancers, Psychological disorders and even neurodegenerative diseases. Based on the data from American Lung Association, asthma is the third-most common cause for hospitalization of children under the age of 15, and the Centers for Disease Control (CDC) has estimated that it affects 6.8 million kids in the U.S. [2]. In addition, over 500,000 people in the United States acquire sepsis each year with a 40% mortality rate and at a cost of $16 billion. Difficult to diagnose and treat, sepsis is typically caused by blood-borne infections inducing a wave of systemic inflammation. This acute inflammatory response can have devastating consequences to host tissues, leading to organ failure and subsequent death. None of the numerous phase 3 clinical trials testing

potential single-target therapies has resulted in a viable treatment [2]. Recently, scientists have found that all of the above-mentioned diseases are more or less related to inflammation. Hence, it is one of the reasons why we should look at this modality, as it has proved that we can ameliorate the systematic inflammation by vagus nerve stimulation from several reports. Additionally, from the report of the best-selling drugs of 2013, the total sales are around $75 billion, and almost half of the sales numbers are directly related to inflammatory diseases, so, without doubt, it will be a promising market to enter with an alternative from pharmacy [3]. In fact, there is already at least one startup working on a clinical trial for this market, which I will mention in the later part. On the other hand, there are several issues that implantable or portable neuromodulation device will be superior to the traditional pharmaceutical medication. First, with the outrageous usage of antibiotics for microorganisms and pathogens, superbug’s problems are appearing around the world, and World Health Organization (WHO) has made several recommendations on this issue [4]. If we use neurmodulated techniques, it is possible that we can circumvent this issue as we implement our own immune system, but it needs more research to back this notion. Second, even though the medication is powerful, somehow people’s adherence to medication is not so good, especially for the patients at home with chronic diseases, which can be improved greatly, if we design a robust and close-loop implantable or portable device. By and large, the above examples and information can bolster that this novel method is worth to dig into for the wellness of human beings. In the next paragraph, I will start from the review of the underlying mechanism of these two systems.

II. Review of the underlying Physiology and Pathology [5] – [11] In these two decades, lots of evidence offers strong support for various interactions among the central nervous system (CNS), peripheral nervous system (PNS) (both sympathetic and parasympathetic branches), the endocrine system, and the immune system as Fig. 1. Inside these complex systems, neurohormonal pathways use cortisols and catecholamines, including norepinephrine, as the controlling and communicating agents, such as the hypothalamic-pituitary-adrenal (HPA) axis utilizing the anti-inflammatory effects of glucocorticoids as Fig.2, the hypoththalamic-pituitary-gonadal (HPG) axis through sex hormones and hypothalamic-pituitary-thyroid (HPT) axis. Though neurohormones in the circulation regulates the immunity at a system level, through neurohormonal binding to receptors in immune cells, this neural pathway regulation of immunity gives an added dimension to immune regulation at a local and regional level. This kind of organizational design let each immune organs be specialized to regulate different aspects of immune function. For example, lymph nodes and thymus regulate cellular immunity (T lymphocyte developing and maturing); spleen and bone marrow regulate humoral immunity (B lymphocyte maturing); mucosa and skin provide the front line defense cells of innate immunity. The nervous system has several characteristics that make it an ideal partner with the innate immune system in immediate nonspecific host defense. It reacts rapidly (in the order of milliseconds to minutes) to many types of nonspecific environmental stimuli. Neurotransmitters and neuropeptides often bind to G-protein-coupled receptors that activate the same secondary signaling pathways (such as those including protein kinase A, cyclic AMP and protein kinase C) as signals triggered by immune mediators.

Through innervation of the immune organs like the lymph nodes, thymus and the spleen, the sympathetic nervous system (SNS) controls immunity at a regional level, and it has both pro-inflammatory effect (redistributing immune cell population acutely) and anti-inflammatory effect (releasing massive norepinephrine (NE) under stress condition). Clinical studies also suggest NE is involved in wound healing. The PNS regulates immunity at sites of inflammation, wherever in the body this might occur. Neuropeptides released from peripheral nerves tend to be pro-inflammatory and are largely responsible for the characteristic features of ‘‘calor, rubor and dolor” (heat, redness and pain) at inflammatory sites. The parasympathetic nervous system has been shown to play a crucial role in immunomodulation. Both afferent and efferent parasympathetic activity is thought to play a role in immunomodulation. Hence, the following contents will focus mainly on this system. By the nature of its ‘‘wandering” route through the body the vagus nerve may be uniquely structured to provide an effective early warning system for the detection of pathogens as well as a source of negative feedback to the immune system after the pathogens have been cleared. The vast majorities of vagal fibers (upwards of 80%) are sensory in nature and thus provide an effective coverage of the body for the detection of invaders. It has been shown to have connections to the heart, lungs, liver, and esophagus among other organs. Additionally, the afferent vagus nerve has interleukin (IL)-1 receptors expressed by paraganglia cells located in parasympathetic ganglia. The presence of cytokines such as IL-1 in the periphery is relayed via the vagus nerve to CNS structures, one of the most important being the nucleus of the solitary tract (NTS). Ascending from the NTS the vagus reaches the parabrachial nucleus, the thalamus, the paraventricular nucleus, the central nucleus of the amygdala, the insula cortex, and in animals the infralimbic cortex including the homologus sites in humans of the anterior cingulate cortex (ACC) and the medial prefrontal cortex (MPFC). At the NTS the afferent and efferent aspects of the parasympathetic nervous system meet. Therefore the NTS is a major relay station for neural-immune communication. On the afferent side, vagal afferents terminate in the NTS in a somatotopic manner resulting in functional divisions of the NTS. This somatotopic organization may allow for a high degree of localization and specificity of immune-to-brain communication. This is important as the anatomical location of the pathogens conveys information necessary to mount a location specific and thereby more effective response. Besides, the NTS has direct and indirect connections to a wide range of neural structures thus giving the vagus nerve the capacity to influence a broad array of processes. On the efferent side, the NTS provides input to the dorsal motor nucleus of the vagus (DMV) and the nucleus ambiguous (NA). These are the sources of the efferent signals, which innervate many of the organs associated with the immune system including the heart, liver, and gastrointestinal system. Acetylcholine release from the vagus nerve modulates immune responses at least in part via alpha 7 nicotinic receptors that inhibit NF kappa B and thus cytokine synthesis and release. The source of the regulatory acetylcholine is suggested that it may be immune-cell derived instead of being released from nerve endings. Taken together these parasympathetic pathways form what has been termed ‘‘the cholinergic anti-inflammatory pathway” as Fig.3. The classical idea that Sir Henry Dale put forward that each neuron had only one neurotransmitter associated with it, (i.e., the post-ganglionic sympathetic neurons used norepinephrine exclusively whereas the post-ganglionic parasympathetic neurons used

acetylcholine exclusively) has been shown to be incorrect. Thus neurons may release a number of substances that may serve to modulate each other and thus produce a complex range of effects. This process has been called co-transmission. For instance, specifically it was demonstrated that neuropeptide Y (NPY), which is co-released from sympathetic nerves with NE, has both inhibitory effects via alpha-adrenergic receptors at low concentrations of NE and stimulatory effects via beta-adrenergic receptors at higher concentrations of NE as indexed by IL-6 secretions of spleen slices. In clinical studies, one group examined the relationships among vagal function as indexed by 24 h heart rate variability (HRV), sympathetic activity as indexed by overnight urinary norepinephrine (NE), and inflammation as indexed by C-reactive protein (CRP) and white blood cell counts (WBC). Another group had a result suggests that vagal function is important in the regulation of both acute and chronic inflammation in healthy humans from acute stressor experiments. Moreover, in humans, a group found that the inverse association between HRV and CRP was 4.4 times greater in females than in males. Consequently, many factors need to consider when we design an experiment. The cholinergic anti-inflammatory pathway literature results are listed at table 2. At the last of this section, I integrate the potential biomarkers options for us to monitor. There are many neuroendocrine and neural factors with component of the innate immune system as table 1. In addition to IL-1𝛽, 2, 6, 8, 12, 17, 18, MCP-1, interferon-𝛾 and tumor necrosis factor-𝛼 (TNF) as general innate cytokines, high mobility group box 1 (HMGB1), a protein released by necrotic cells, and activated macrophages and somatic cells during severe sepsis, is also a common pro-inflammatory cytokines. HMGB1, which is secreted later than the ‘classic’ pro-inflammatory cytokines (12–18 hours after administration of Lipopolysaccharide (LPS) in vivo compared with TNF and IL-1, which peak at 2 hours and 4–6 hours, respectively. On the other hand, anti-inflammatory cytokines panel has IL-4, IL-10, IL-13 and transforming growth factor-𝛽. Lastly, there are Toll-like receptor (TLR) 2,3,4,7 and 9 that will respond to various pathogens and turn on the transcriptional factor nuclear factor (NF)-𝜅B. Next paragraph I will propose a system in order to let us understand more clearly about these two systems’ mutual effects.

Table 1 Neuroendocrine and neural factors with component of the innate immune system

(Abstracted from [5])

Table 2 Experimental disease models responding to the cholinergic anti-inflammatory pathway. [Abstracted from [8]] III. Proposed System

In order to study these two complex systems, we need a platform that can continuously record and stimulate the nerve, and monitor those biomarkers from body fluid. Although I expect that with the continuing progress of Micro Electro-Mechanical System (MEMS), System on a Chip (SoC) and System in Package (SiP), one day, we can manufacture this all in one system with the specification I mentioned, I will adopt separate devices for this current design, as SiP just got its momentum to grow and BioMEMS still has no united industry standard for its process yet, especially microfluidics. With the former section’s introduction, we know that many diseases are originated from inflammation that break the earlier homeostasis or aggravated the symptoms with inflammation or immune suppression. Hence, the biomarkers of the immune response will be our targets, such as those cytokines and other molecules that I list at the last paragraph of the former section. Nowadays, the gold-standard testing protocol will use blood (venous or capillary blood), interstitial fluid or urine and then, after processing the sample, run the Enzyme-Linked Immunosorbent Assay (ELISA), which has quite nice sensitivity and specificity in general, for the analytes. It will be a nice option if we only want to test few objects. But if we want to test a panel with more than twenty analytes, the procedures will take much time, need expensive instruments and use many reagents to complete the test. Fortunately, with the development of mircrofluidics in these two decades, now we have the technique to miniature the whole procedures on a tiny microfluidic chip. I adopt Self-powered Integrated Microfluidic

Blood Analysis System (SIMBAS) for the diagnostic part [12]. Furthermore, I want to streamline and simplify the whole procedures more by improving the first procedure, collection of samples, so I would like to modify my former design [13], a microneedle array with a diagnostic platform for hypo- and hyperthyroidism as Fig 4, to match the current specification, immune biomarkers. Fortuitously, there is a HPT axis that will affect the immune response too, so I can keep the Thyroid-stimulating Hormone (TSH), Free T3 and Free T4 testing channels, and expand other channels for other testing analytes. On the whole, it can greatly lower the cost and operating time.

+ Fig. 4 Schematics of the proposed system, a microneedle array combines a diagnostic platform. [Abstracted from [12] [13]] On the other hand, because we want to decipher the hidden pattern that responds to certain immune conditions, we need an invasive and implantable device for a better signal-to-noise ratio (SNR) to continuously record the spatio-temporal signal at the afferent vagus nerve around cervical portion or around NTS, which might be riskier in the operation, for the place is closer to the brain stem. Scientists believe there is a map for the neuro-immune somatotopic code [14]. Therefore, we can design an experiment delivering same extents of damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) at different body part and see whether there is any concealed clues inside the recording pattern; it is possible that we can utilize the machine learning technique, unsupervised learning method, to help us sort out and cluster the massive data. On the contrary, for the therapeutic part, currently there are emerging fashions that are less invasive or almost non-invasive for vagus nerve stimulation, which we can use with possibly better outcomes after we figure out the pattern for specific responses, and usually it will be easier for people to embrace this technique as it has lower risk than the implantable solution. But, of course, it will need to be researched and confirmed that whether they can gain the same results as the implantable solution; several ideas are currently under clinical trials and I will mention some in the analytic section. There are various proposed systems for VNS reviewed in [2]. I adopt ADNS-300 as our platform, since it combines both recording and stimulation functions with a long battery lifespan as Fig.5. In the experiment, I will record both side of vagus nerves, but only stimulate left vagus nerve, where has less efferent nerve to cardiac branch. Next section  I will analyze from different perspectives for the potential applications.

Fig. 5 A real product of the stimulator and schematic representation of the ADNS-300 electrode, reference to [2]. IV. Prioritized Analysis

First of all, we should look at the current neumodulation market if we take it seriously to push forward the idea into the clinical space. So far, there are 4 major companies that are leading in this segment, Medtronic® (M), Boston Scientific® (B), St. Jude Medical® (SJ) and Cyberonics® (C). M is still the leader with dominated revenue comparing to others, by the use of its heart electrical stimulation expertise (pacemakers and defibrillator) to treat nervous system-related diseases and conditions. They invested much to explore multiple uses with their technology, including movement, chronic pain and psychiatric disorders by gastric electrical stimulation, spinal cord stimulation or sacral neuromodulation. Over the past years, its deep brain stimulation obtain quite success at Parkinson’s disease treatment, but they seems less interested in VNS. B will be the number 2 in this segment, traditionally focusing on treating neuropathic pain with its Precision spinal cord stimulation system. The Precision Spectra is outfitted with 32 contacts and four lead ports, which is also in the lead to comparing products. They are also interested in the application of Parkinson’s disease and have a nice data from a previous trial. However, they flop to demonstrate clinical benefit of VNS in heart failure on August 24 conference. The revenue of SJ is very close to B, but its projection is not as promising as B. SJ had issues with FDA years ago, so they try to come back to this segment recently with its spinal cord stimulation system for chronic pain treatment using burst stimulation instead of the standard tonic stimulation, billed using 95% less energy than others. Last one, C is well known for its VNS system for refractory epilepsy and treatment-resistant depression, and C is assessing autonomic regulation therapy for patients with chronic heart failure as B with a better clinical result but it was less convinced as it did not have control group [15]. The above 4 companies will possibly have more resource to invest in this emerging field and push forward the preclinical results into clinical usages. Besides from these big companies, a startup, SetPoint Medical, backed by Boston Scientific utilizing neuromodulation therapies for patients with inflammatory autoimmune diseases and they have a nice feedback from its ongoing clinical trials for rheumatoid arthritis (RA). In addition to the biomarkers I listed before, there is another group of cytokines, we should also consider, the IL-20 subfamily, which also interferes the host defense and homeostasis, including IL- 19, 20, 22, 24 and 26. This subfamily mainly facilitates the communication between leukocytes and epithelial cells, so certain diseases should take them into account like psoriasis, inflammatory bowel disease (IBD), liver inflammation RA and certain cancers as they activate STAT3, which is a well-known oncogenic transcription factor with an imperative roles in many different types of cancers [16]. The most important discovery in this field in these two decades should be the cholinergic inflammatory reflex proposed by Dr. Tracey’s group, who is also the co-founder of Setpoint Medical. The reflex can help us attenuate the systemic inflammation [17], so it should be possible to propose a combo therapeutic method that combines the reflex with local and regional immune modulation to expand the potential applications to more aspects. Apart from cholinergic and former neurohormonal pathways, there was a new catecholamine pathway was discovered last year, using dopamine to mediate vagal modulation of the immune system by electroacupunture of the sciatic nerve at ST 36

Zusanli acupuncture point. This mechanism also controls systemic inflammation with the production of dopamine in the adrenal medulla [18]. The manner will be favorable as it utilizes less invasive method to modulate the VN. Nevertheless, it should belong to the humoral immune pathway, which will not responds as fast as the cholinergic reflex pathway. Additionally, in recent years, there are many reports about transcutaneous vagus nerve stimulation (t-VNS) via the auricular branch of the vagus nerve of the left tragus. If it is proved to be as influential as direct VNS, the best modality will be integrate t-VNS and electroacupuncture at the sciatic nerve into a portable system as it will be able to modulate both of the parasympathetic and neurohormonal pathways. As for now, since we need to have more evidence and information to support this notion, I will still adhere to the implantable solution in order to obtain better signal, analyze the pattern for the behind mechanisms, explore novel neural circuit from the integrated results of A𝛿 and C fibers at the afferent part of VN fibers and A𝛿,A𝛽 and C fibers at the efferent part, and last establish a more solid foothold. Many diseases have been studied with VNS as I listed in the former contents. Consequently, I am not interested so much in them; I would like to research VNS at other opportunities that should be able to ameliorate the symptoms or cure the diseases from the point of view on systemic inflammation: neurodegerative diseases (Alzheimer’s disease), allergic responses (asthma) and certain cancers. No matter we look at the viewpoints from patients and patients’ family or the lucrative market values, it undoubtedly will be reasonable to take a shot for this potential new route as there hasn’t been a really nice treatment for each disease so far. If the result is promising, we might offer a more proactive alternative to prevent these diseases from exacerbation or even happening. Or it is possible to design a combo treatment with medication. One possible scenario will be that as drug development sometimes used to target specific cytokine, for diseases affecting multiple cytokines might not get the result as influential as we expected, so if we can use VNS to modulate the systemic inflammation with the medication targeting the specific pathway, it will be possible to boost the effects or counteract the side effects from the medication. In the following, I will briefly go through why it is feasible to use VNS to treat them. The inflammatory response during most chronic neurodegenerative diseases is dominated by the microglia and mechanisms by which theses cells contribute to neuronal damage and degeneration. Microglia are primed by prior pathology or by genetic predisposition and respond more vigorously to subsequent inflammatory stimulation, thus transforming an adaptive CNS inflammatory to systemic inflammation, into one that has deleterious consequence for the individual [19]. If we can reproduce the similar protective results that long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) against Alzheimer’s disease (AD) and Parkinson’s disease (PD) by VNS, then we can greatly substantiate my viewpoint, because the mechanism can be modulated by either humoral pathway or reflex pathway. Probably it will be not simple to reverse fully but it has great potential to prevent exacerbation of the progression of disease. Another advantage of VNS is that it won’t cause immunosuppression as medication does, at least from current review’s opinions. The endpoint of this experiment will be comparing the patients with control groups for the traditional tests plus the portfolio of biomarkers: interferon- 𝛾  (IFN− 𝛾), IL− 1𝛽, IL− 4, IL− 12, IL− 13, IL− 10  or  CD200,   TNF- 𝛼 ,

inducible nitric oxide synthase (iNOS), TGF-𝛽, glucocorticoid, prostaglandin, CX3CL1 and CCL2. Energy metabolism, obesity, and genetic instability are regulated in part by the relationship of the organism with commensal bacteria that affect inflammation with both local and systemic effects. Different aspects of inflammation appear to regulate all phases of malignant disease, including susceptibility, initiation, progression, dissemination, morbidity, and mortality. Besides, aspirin or other NSAIDs also have effects to decrease incidence of tumors, so it is supportive that there is a role for inflammation in cancer susceptibility. NF-κB and STAT3 are among the best characterized of these transcription factors in cancer. NF-κB induces many genes regulating the cell cycle, angiogenesis, and cell survival. It also induces NOS2; COX2; and most genes encoding cytokines, chemokines, and proteases. In different experimental cancer models, for example in liver and colon carcinogenesis, NF-κB expression is required in both the tumor cells and the infiltrating hematopoietic cells [20]. All of the information shows us VNS can be a wonderful candidate to combat specific cancers. The endpoint of this experiment will combine the traditional imaging test plus the portfolio of biomarkers: TGF-𝛽, IL-6, IL− 1𝛽, IL− 1𝛼, IL-4, IL-8, IL-10, IL-11, IL-12, IL-13, IL-17, IL-18, IL-20 subfamily, IL-23, CCL2, CRP, prostaglandin, EGFR, VEGF and fibroblast growth factor 2 (FGF2). Asthma is a debilitating respiratory disease characterized by exaggerated bronco-constriction and the associated symptoms of wheezing and shortness of breath. Importantly it is thought that inflammation of the airways is the major precipitating factor; the parasympathetic control of the airway is important for an understanding of asthma. There is an evidence of cotransmission of acetylcholine and nitric oxide (NO) at cardiac cholinergic nerves. This type of co-transmission may be important for the regulation of bronchial tone as acetylcholine is implicated in bronco-constriction whereas NO is implicated in bronco-dilation. The increasing incidence of asthma in children may be caused by the widespread use of broad-spectrum antibiotics that may modify the commensal flora and the development of the immune system [7] [21]. Combining these information, as usual, VNS will still be a fantastic candidate for asthma treatment, though I will prefer adopting non-invasive or less-invasive methods after we confirmed the result from implantable method at preclinical study as, generally speaking, people won’t accept the procedure only for asthma. The endpoint for this experiment will combine the traditional tests such as skin prick test (SPT) positivity, PC20 and FEV1/FVC plus the portfolio of biomarkers: CRP, IL-4, IL-5, IL-6, IL-13, IL-17, IL-25 (main target), IL-33, CCL5, CCL11, CCL17, CCL22, CCL24, thymic stromal lymphoprotein (TSLP), MUC5ac, IFN− 𝛽, IFN− 𝛾 and immunoglobulin E (IgE). Lastly, serendipity will be Type 2 diabetes as it also uses NF-κB pathway. The protocol for all of the above experiments will use my proposed system to study these diseases, if the result promises well, we can consider the usage of the non-invasive or less-invasive tools to see whether we can get the similar result; then we can continue the following phases of clinical trials. The testing platform for biomarkers will check all of the markers I list in section III, in order to gain more input parameters for future analysis; it is also a critical phase for the entrance of the tools from machine learning or deep learning. We will build a model, give parameters to each input feature depending on the correlation with the disease, and then pick up the candidates that were not find out in the past into the final experiment with the portfolio I mentioned for each

disease. Possibly we can also optimize a generalized diagnostic model for the future application of automatic diagnostics; the stimulated parameter can also follow the same procedures to be optimized for a best therapeutic results. All in all, I believe now is really an exciting and promising time for the neuromodulation field; there are a lot of objects that we can explore deeply with current or developing tools, and one day we will figure out the mysterious puzzle between nervous system and immune system and it will be possible that we can invent a new vaccine fashion by these discoveries.

Fig. 1 Schematic illustration of connections between the brain and immune system. [This figure is abstracted from [4]] Fig. 2 Effects of glucocorticoids on Fig. 3 The inflammatory reflex immune-cell populations. choline acetyltransferase-expressing T IFNγ, interferon-γ; NK cell, natural killer cells (ChAT+ T cells), β2AR, β2- cell; TC, cytotoxic T cell; TH, T helper cell. adrenergic receptors. [This figure is abstracted from [5]] [Abstracted from [11]]

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