Wandering between Two Worlds: A Closer Look at the Neuroimmune Communication

Do you pass out when you see blood? Have you ever been so emotionally shocked that the next thing you know, you are lying on the floor surrounded by people asking if you are all right? Fainting is a consequence of direct stimulation of the vagus nerve. This nerve is often termed, the “wandering” nerve, because as it leaves the brainstem, it “wanders” through organs in the neck, thorax, and abdomen. This means that the vagus nerve regulates various tasks such as heart rate, swallowing, speech, and breathing – tasks that are vital for our existence. Most of us likely know someone suffering from hypertension and the cardiac diseases associated with high blood pressure. However, low blood pressure can be as equally dangerous because it can lead to stimulation of the vagus nerve, which causes the blood flow to be redirected from the brain to the heart and the rest of the body. Such a temporary lack of blood flow and oxygen to the brain triggers vasovagal syncope, a fancy term for what we call fainting. During emotional shock, there is an increased stimulation of the vagus nerve, which in turn leads to slowing of the heart thereby causing a blood pressure drop and hence, fainting. Stimulation of the vagus nerve does not only result in fainting, but it also triggers vomiting. If you are an EMT, this is the reason why an oropharyngeal airway (OPA) must only be used on unconscious patients without a gag reflex. Insertion of the OPA in the back of the pharynx may stimulate the vagus nerve and result in vomiting, thereby presenting an airway obstruction.

This wandering nerve is not only involved in the physiological tasks that are vital for us, but it has also been implicated as an immunomediator thereby presenting a linkage between the nervous and the immune system. Research aimed towards understanding the mechanisms behind neuroimmune communication has been gaining popularity in recent years. For instance, receptors for the neurohormone, melatonin, a regulator of the circadian rhythm, has been found on human T cells, hence giving melatonin regulatory functions in the immune system as well (Lardone et al. 2011). Likewise, cytokines secreted by immune cells also control neural and glial activity in the brain such as neuroplasticity, a phenomenon that takes place during learning and memory where new neural synapses are formed or reorganized (Huh et al. 2000). Furthermore, major histocompatibility complex (MHC) proteins, which allow antigen presentation in the immune system, are also found in neurons (Trakhtenberg and Goldberg, 2011). If cytokines released by immune cells can regulate neuronal function, can other neurotransmitters released by neurons also regulate immune function?

Recent findings by Rosas-Ballina et al. (2011) reveal that the vagus nerve plays a key role in orchestrating the neuroimmune communication. The vagus nerve stimulates celiac ganglion adrenergic neurons, which in turn, can stimulate the spleen. The celiac ganglion is basically a collection of neurons located in the upper abdomen that can innervate the spleen, stomach, liver, gallbladder, kidney, small intestines, and the colon. These neurons are termed adrenergic because they have receptors that can interact with norepinephrine or noradrenaline. This pathway eventually leads to the regulation of TNF-α release by splenic macrophages. Furthermore, activation of these macrophages depends on the neurotransmitter, acetylcholine (ACh), and activation of nicotinic ACh receptors (nAChR) on splenic macrophages. However, there is a paradox in this pathway because the nerve fibers that innervate the spleen are adrenergic and they release norepinephrine as their primary neurotransmitter. Hence, where did the ACh that innervated the macrophages come from?

Rosas-Ballina et al. (2011) show that it is not neurons that are secreting the ACh responsible for activating macrophages. Rather, it is splenic T cells that release ACh after being innervated by the splenic nerve fibers. These T cells express adrenergic receptors that can respond to norepinephrine secreted by the splenic nerve fibers. Hence, T cells are essentially functioning like neurons that can respond to neuronal signals and release a neurotransmitter in response! Using a specific strain of mice called BALB/c, the authors found that electrical stimulation of the vagus nerve in these mice increased acetylcholine levels within minutes, as measured by microdialysis and mass spectrometry. Microdialysis is simply a semi-invasive technique that allows researchers to measure the amount of neurotransmitters or other molecules that are transported to or generated within the extracellular milieu in virtually any organ in the body. This technique is especially useful in in vivo studies and relies on diffused-based separation that allows molecules to diffuse through a semi-permeable dialysis membrane. Next, the authors determined which cells are releasing the ACh since the splenic nerves are releasing norepinephrine as their primary neurotransmitter. The authors saw that adrenergic splenic neurons terminate at the white pulp region of the spleen that contains T cells. Therefore, they hypothesized that perhaps, these T cells could be the cells responsible for secreting ACh. In order to test this hypothesis, the authors incubated splenic T lymphocytes in the presence of norepinephrine and found that norepinephrine increased ACh production by these T lymphocytes, suggesting that stimulation of adrenergic splenic neurons may trigger the release of ACh by splenic T lymphocytes. In order to show that T cells communicate with macrophages via ACh in order to mediate inflammation, the authors stimulated the vagus nerve of nude mice, which lacked functional T cells and are therefore, unable to respond to splenic nerve fibers. Results show that vagus nerve stimulation in the nude mice during endotoxemia (the presence of endotoxins in the blood, which should theoretically stimulate the immune system) failed to decrease production of TNF-α by macrophages , as measured by Enzyme-linked immunosorbent assay (ELISA). Simply, ELISA determines the amount of antibody or antigen present in the sample. In this case, the cytokine, TNF-α, was tethered to the bottom of the plate and a specific antibody was added unto it to detect its presence. The results of this experiment are in contrast to the findings obtained from control mice, which had functional T cells, where vagus nerve stimulation during endotoxemia attenuated TNF-α production.

The authors then identified the subpopulation of T cells that mediate this response by using a ChAT(BAC)-EGFP mice, which express EGFP, a fluorescent protein, under the promoter for ChAT, an enzyme required for ACh biosynthesis. They found that ChAT-EGFP was largely expressed in CD4+ T cells. The co-localization of the splenic nerves and T lymphocytes were also assessed by immunofluorescent micrographs in order to detect whether the splenic nerves does indeed directly stimulate the T cells. Immunofluoresent micrograph involves a process that attaches fluorescent secondary antibodies to primary antibodies, which in turn attaches to the antigen of interest. Once light illuminates the fluorescent secondary antibody, it emits a light that the investigator can photograph. Immunofluorescent micrographs of spleen sections show that ChAT-EGFP expression in T cells was adjacent to the splenic nerve fibers that are expressing a specific enzyme called tyrosine hydroxylase, which is crucial for norepinephrine biosynthesis. Further examination of the anatomical basis shows that synaptophysin, a glycoprotein found at neural synapses, was adjacent to the CD4+ T cells in the white pulp, suggesting that synaptophysin-positive nerve fibers terminate at the CD4+ T cells. Therefore, the norepinephrine released by these splenic nerve fibers can innervate the T cells present in the white pulp to release ACh. Indeed, analysis of mRNA levels of adrenergic receptors that may respond to norepinephrine were found on the T cells, further providing evidence that the adrenergic receptors on T cells underlie the mechanism of splenic nerve signaling. It should be noted that the authors discovered the presence of CD4+ T cells containing ChAT not only in the spleen, but also in the lymph nodes and Peyer's patches, which possibly implicates this signaling pathway in these regions as well.

The authors further tested their theory in vivo by transplanting CD4+ T cells, which express adrenergic receptors and contain the enzyme ChAT required for ACh biosynthesis, into nude mice, which lacked a functional T cell repertoire. Transplanting these T cells into mice devoid of T cells during endotoxemia decreased serum TNF-α levels after electrical stimulation of the vagus nerve. These findings are in contrast to those observed in the control in which the nude mice received CD4+ T cells that did not contain ChAT, suggesting that this enzyme is indeed required for ACh biosynthesis and hence, suppression of TNF-α production. Furthermore, the authors discovered that the CD4+ T cells containing ChAT significantly accumulated around splenic nerve fibers containing synaptophysin, which validates their anatomical theory that splenic CD4+ T cells interact with splenic nerve fibers. The role of ChAT in the mechanistic pathway was further examined by knocking down ChAT in spleen CD4+ T cells using small interfering RNA against ChAT. These ChAT siRNA-transfected CD4+ T cells were then adoptively transferred into nude mice. Results show that after electrical stimulation of the vagus nerve, the mice that received the CD4+ T cells transfected with ChAT siRNA failed to decrease TNF-α production by macrophages. Therefore, to summarize the pathway that mediates immunosuppression, stimulation of the vagus nerve activates the celiac ganglion adrenergic neurons to release norepinephrine, which can then act on adrenergic receptors present on CD4+ T cells. Activation of these receptors on CD4+ T cells innervates those T cells to produce ACh, which can in turn act on nicotinic ACh receptors on splenic macrophages to decrease their production of TNF-α, thereby leading to immunosuppression as shown in the Figure.

The findings of this study implicate the role of the vagus nerve in mediating the degree of immune response in addition to its already established role in regulating the heart rate, blood pressure, and other vital physiological process. This study also conducted both in vitro and in vivo experiments, which further validate their hypothesis and findings because they showed that their results were replicable not only in cells but also in live animals. Most importantly, the results of this study offer promising treatments for inflammatory diseases, such as rheumatoid arthritis, by means of manipulating the vagus nerve. This manipulation could comprise of medical devices or pharmacologic agents that promote stimulation of the vagus nerve in order to suppress the inflammatory response. This study also raised several questions about the specific mechanism regarding neuroimmune communication. For instance, is it specifically the ACh produced by the T cells and the norepinephrine produced by the splenic nerve fibers that mediate the observed immunosuppression or do other subpopulations of immune cells also participate in the process? In addition, CD4+ T cells containing ChAT, required for ACh biosynthesis, have also been found in the lymph nodes and Peyer's patches. It would be interesting to find whether the CD4+ T cells in these regions also behave in the same manner or have the same mechanisms as those obtained from this study in the spleen. This would certainly be worth pursuing in future studies to further unravel the cross talks between the nervous and immune system.

Works Cited

Huh, G. S., et al. "Functional Requirement for Class I MHC in CNS Development and Plasticity." Science (New York, N.Y.) 290.5499 (2000): 2155-9. Print.

Lardone, P. J., et al. "Melatonin Synthesized by T Lymphocytes as a Ligand of the Retinoic Acid-Related Orphan Receptor." Journal of pineal research 51.4 (2011): 454-62. Print.

Rosas-Ballina, M., et al. "Acetylcholine-Synthesizing T Cells Relay Neural Signals in a Vagus Nerve Circuit." Science (New York, N.Y.) 334.6052 (2011): 98-101. Print.

Trakhtenberg, E. F., and J. L. Goldberg. "Immunology. Neuroimmune Communication." Science (New York, N.Y.) 334.6052 (2011): 47-8. Print.