Neural modulation of immune function

Journal of Neuroimmunology, 10 (1985) 59-69

59

Elsevier JNI 00324

Neural Modulation of Immune Function Thomas L. Roszman and William H. Brooks Departments of Medical Microbiology, Immunology and Pathology, University of Kentucky Medical Center, Lexington, K Y 40536 -0084 (U.S.A.)

(Received14 May, 1985) (Revised, received26 June, 1985) (Accepted 26 June, 1985)

Summary In this report we review our hypotheses and approaches to the study of the relationship between the central nervous and immune systems. Discussed are results pertaining to the modulation of immune parameters resulting from perturbations of the brain employing electrolytic lesions and the neuroleptic 6-hydroxydopamine. Experiments describing the central and peripheral effects of serotonin on in vivo and in vitro immune responses are also discussed. Key words: 6 - H y d r o x y d o p a m i n e - I m m u n e r e s p o n s e - N e u r a l lesions - S e r o t o n i n

Introduction Neuroimmunology is a rapidly evolving branch of neuroscience that attempts to investigate normal and pathological central nervous system (CNS) development and function using a variety of immunological probes and techniques, e.g., search for normal and neoplastic glial surface antigens; establishing specific neurotransmitter pathways. Additionally, those changes in immunological reactivity that occur in various neurological diseases, e.g., multiple sclerosis, are determined in an attempt to correlate altered immune function with CNS pathogenesis. Recently, the quest to establish a relationship between the C N S and the immune system has been more sharply focused to address the hypothesis that in addition to its known regulatory functions the CNS also is capable of modulating immune reactivity. Conversely as in This work was supported in part by NIH Grants NS 17423 and CA 18234. 0165-5728/85/$03.30 © 19~ F.lsevierSciencePublishers B.V. (BiomedicalDivision)

60 other neural feed-back loops, the brain should be influenced by the generation of an ongoing immune response. Conceptually, neuroimmunomodulation (NIM) is very appealing to neuroscientists yet has not been enthusiastically embraced by the immunological community as a whole. Current immunological doctrine states that the immune response is autologous and self-regulating, consisting of antigen-driven T-helper and T-suppressor cell systems (Green et al. 1983), idiotype and anti-idiotype networks (Jerne 1974) and genetic controls (Benacerraf and McDevitt, 1972). These self-contained networks considered with the fact that immune responses can be elicited in vitro (thereby escaping any hypothetical link with the CNS) as well as the difficulty in imagining that the specificity of lymphocyte receptors can be modulated by neurally derived 'immunomodulatory transmitters' form the basis of the arguments against NIM. Despite these objections, data continue to accrue indicating that a functional link between the CNS and the immune system does exist. Thus, the concept of NIM can be neither ignored nor summarily dismissed. Assuming these data to be correct, the question no longer turns on a simplistic demonstration of interrelationships between the CNS and immune responses but rather what is the nature of these complex interactions. Admittedly, much of the data supporting this network indeed is demonstrative, yet, as in all new fields of scientific endeavor, one must initially set the limits for investigation by describing the phenomena. This having been achieved, the stage is presently set for a more mechanistic approach. Our approach to the study of NIM is based on 2 hypotheses which may explain the immunomodulatory role of the CNS. The first suggests that the nervous system independently regulates immune reactivity prior to any and all immunological events. Thus, the CNS 'sets the gain' of immunological reactivity by supra-segmental changes in either (or perhaps both) central and peripheral neurotransmitter pathways or neuro-endocrine networks and, subsequently, the innervative a n d / o r hormonal milieu of the immune system, thereby regulating the response before antigenic stimulation. Such pre-existent interrelationships would explain the immunosuppression observed during stress and bereavement (reviewed by Stein 1985). Furthermore, similar neuromodulation offers an explanation of the variation observed in normal individuals sequentially tested by a variety of immunological probes (personal observations). Whereas the first hypothesis implies a considerable, if not absolute, degree of autonomy of the CNS in regards to its regulation of immune readiness, the second thesis suggests the presence of a balanced neuroimmunologic network. Accordingly, as immunological reactivity increases, flux in neurotransmitters in specific areas in the brain occurs. This activity, in turn, may result in alterations in autonomic nervous system and neuro-endocrine activity which effectively alter lymphocyte function through interplay with a specific neurotransmitter a n d / o r hormonal membrane receptor(s). Support for this hypothesis is gained from the observations that hypothalamic activity is increased during the peak of immune responsiveness (Besedovsky et al. 1977) as well as those showing that the levels of norepinephrine in these regions dramatically change following immunization (Besedovsky et al. 1983).

61 Therefore, an efferent/afferent neuroimmunologic pathway is hypothesized where the CNS modulatory influence is triggered via an on-going immune response thereby allowing the CNS to act as a governor of immunological responsiveness. We have approached the concept of NIM by employing differing experimental models. Initially, we employed classical cerebral mapping techniques in efforts to explore various areas of the brain that may be influential in immune function. Having demonstrated that specific areas of the brain are associated with suppression and facilitation of lymphocyte function, we are currently exploring the role of central and peripheral neurotransmitters in mediating CNS influence of immunological function. In the following, we will review our findings with emphasis on the utility as well as pitfalls of these approaches.

Neural Lesions and Immune Reactivity

Our initial studies were directed to confirming the earlier reports that anterior hypothalamic ablation is associated with diminished immune reactivity (Stein et al. 1976). In these studies we demonstrated that electrolytic lesioning of the anterior hypothalamic areas (AHT) of rats induces a decrease in the numbers of nucleated spleen cells and thymocytes as compared to control lesioned and normal animals (Cross et al. 1980). This modification extends also to mitogen (Cross et al. 1980) and antigen-driven lymphocyte proliferation (Roszman et al. 1985) and natural killer cell function (Cross et al. 1984). The observed diminished immune reactivity was short-lived; returning to normal within 14 days. Exploration of other areas of the brain indicated that in addition to areas that are associated with down-regulation of immunity there are specific areas (hippocampus and amygdaloid complex) that facilitate lymphocyte reactivity with a marked enhanced proliferative response (Brooks et al. 1982). These effects also disappeared by 14 days following lesioning. The acuteness of the effects are attributed to the remarkable plasticity of the CNS and the subsequent re-establishment of dendritic-axonal networks (Goldowitz et al. 1979). Next, we began to explore what modulatory effects of CNS had on the mediators of immunity. As an initial approach, we fractionated spleen cells obtained from rats with AHT lesions on glass-bead columns to obtain adherent and non-adherent cell fractions (Roszman et al. 1982). Removal of this adherent population results in a significant increase in mitogen-induced reactivity. Further study proved the impairment in responsiveness associated with AHT lesions to result from a macrophage-like suppressor cell. Interestingly, the increased suppression by these splenic macrophages does not result from increased numbers of suppressor cells but rather from a qualitative difference in suppressive capabilities of each macrophage. Thus, macrophages from AHT-lesioned animals are significantly more suppressive than those obtained from control or normal animals. Conversely, the increase in splenic mitogen reactivity of spleen cells obtained from animals with either hippocampal or amygdaloid complex lesion is the result of a decrease in suppressor macrophage-like activity.

62 From these experiments demonstrating that indeed changes in CNS function can be correlated with changes in immune reactivity, we began the search for the functional link between the CNS and mediators of immunity. Two apparent possibilities exist: one through the secretion of hormones via the neurally regulated endocrine system and the other by innervation of lymphoid organs and the subsequent interaction with neurotransmitters. Although alterations in the concentrations of circulating hormones are associated with changes in lymphocyte activity (Filipp and Mess 1969; Beling and Weksler 1974; Snow et al. 1981), the functional significance of this relationship is unclear. We have shown that modulation of immunity by neural lesions is not related to changes in corticosterone (Cross et al. 1980); yet, hormones may be influenced by A H T lesions that are capable of impairing cellular activation. In order to test this hypothesis neurally induced changes in lymphocyte responsiveness were assessed before and after hypophysectomy (Cross et al. 1984). The results obtained from these investigations suggest that the CNS is capable of modulating splenocyte blastogenesis and number via the endocrine system as pituitary removal abrogates both the inhibitory as well as the facilitory proliferative effects of neural lesions. While the concept of NIM may be formally established using this experimental model, the mechanisms responsible for this modulation are less amenable to investigation. For example, it is well established that macrophage-like suppressor cells are found in the spleens of normal rats (Mattingly et al. 1979). Moreover, the suppressive activity of these cells is significantly increased by experimentally induced tumors (Glaser et al. 1975) or with microbial infections (Camus et al. 1979; Mattingly et al. 1979). Whereas it is clear from our studies that ATH lesions may be correlated with a marked effect on suppressor macrophage activity or indirectly through another cell such as the T-cell which may regulate the macrophage activity, it is not possible to state that these changes are specifically induced by alterations in concentrations of circulating non-corticosteroid hormones, neurotransmitters, or alterations in the innervation of the immune system. At present the immune system of the rat is insufficiently elucidated to be of value in exploring the effects of NIM on an immune network. The mouse model is perhaps better suited yet is complicated by the difficulty in the precise placement of lesions that are required to approach NIM by classical lesioning techniques. Recently the role of circadian rhythmicity in immune function has been studied and found to be important in the timing of lymphocyte reactivity (Fernandes et al. 1976). The close proximity of A H T lesions that induce decreased lymphocyte activity with the area of the brain effecting alterations in circadian rhythms suggests that at least some of the effects of lesioning may be more related to those induced by changes in rhythmicity rather than a direct link between the brain and immune response. Only by studying lymphocyte responsiveness in animals with A H T lesions at various times during a 24 h period can this issue be resolved. Another area of caution in this animal model is the fact that the induced immunological effects are transient. Although the short-lived effects of these lesions have been attributed to the anatomical plasticity of the CNS, it is perhaps more likely that a functional plasticity occurs within the CNS; hence, other areas of the

63 brain are capable of restoring the normal milieu of the host and thereby the functional effects of the lesions, which are anatomically destructive, are only temporary. Alternatively, these modifications induced by the CNS lesions may be of such low 'intensity' that they are easily compensated for within the normal framework of the immune network. These observations raise 2 important questions as to the usefulness of the lesioning model in elucidating the mechanisms of NIM. The first: if the effects of NIM are indeed extremely transient and easily compensated, are they biologically important? The second: would it not be more appropriate to explore models with a more prolonged effect in order to dissect the individual and collective roles of the CNS and immune system in NIM?

Effects of 6-Hydroxydopamine on Immune Function

In the serach for another paradigm to study NIM we have begun a series of experiments to determine the effect of central depletion of catecholamines on immune function. Initially, we injected 100 /~g of 6-hydroxydopamine (6-OHDA) intracisternally into adult CBA mice rather than peripheral injection of 6-OHDA as employed in previous studies (Besedovsky et al. 1979; Miles et al. 1981; Williams et al. 1981). The results of these studies established that animals primarily immunized with SRBC 48 h after administration of 6-OHDA have a markedly impaired primary antibody response as evidenced by decreased numbers of splenic IgM and IgG PFC. Furthermore, we have determined that the immunosuppressive effects of 6-OHDA persist for at least one month. Thus, not only is this form of chemical lesioning easy to perform but it is not acute in nature as are electrolytic lesions. Immunohistochemical analyses reveal that the norepinephrine (NE) cells are confined predominantly to the brain stem; however, the prejections of these neurons are long and diffuse with both ascending and descending innervation patterns (Hokfelt et al. 1984). Using high performance liquid chromatography we determined the concentration of NE, dopamine and serotonin in the brain stem and hypothalamus 48 h after intracisternal injection of 6-OHDA. The results demonstrated that NE was significantly decreased in both the brain stem and hypothalamus as compared to animals injected with saline. Neither serotonin nor dopamine were markedly affected by 6-OHDA treatment. We have examined other aspects of the effects of 6-OHDA treatment on immune function. The results demonstrate that this treatment affects the development of immunologic memory to SRBC but interestingly not the secondary anti-SRBC antibody response. This latter observation suggests that memory cells are refractile to whatever changes occur as a result of intracisternal injection of 6-OHDA, whereas virgin immunocompetent cells are not. Further experiments are required employing protein antigens to extend these observations and to be certain that they are not the result of some peculiarity associated with SRBC antibody response. Having demonstrated that 6-OHDA treatment effects immune responsiveness, additional experiments were performed to examine these immunologic mechanisms. Intracisternal injection of 6-OHDA either 2 h before or 24 h after primary immuni-

64 zation with SRBC does not affect the ensuing primary response. These results indicate that this treatment affects mainly the early events involved in the initiation of the primary antibody response rather than the proliferative and differentiative phases. A number of explanations are possible for the effects of 6 - O H D A on the SRBC antibody response. Because of the influence of hypothalamic-releasing factors on the maintenance and balance of peripherally circulating hormones, it is possible that the effects of N E depletion are related to changes in pituitary release of A C T H and subsequent elevation of potentially suppressive circulating corticosterone. However, blood levels of corticosterone were similarly elevated in both 6 - O H D A - and salineinjected animals yet inhibition of the SRBC response was found only in those that received 6-OHDA. Thus, corticosterone is not responsible for the suppressed antibody responses. Another possible explanation for the effects of 6 - O H D A treatment on the immune response is that it induces qualitative or quantitative alterations in nucleated spleen cell subpopulations. We found neither changes in nucleated spleen cell numbers nor in the percentage of T- and B-cells in 6-OHDA-treated animals as compared to saline-treated control animals. This does not rule out the possibility that more subtle changes in cell subsets are occurring in the 6-OHDA-treated animals. We have in addition examined the antibody response in lymph nodes of 6-OHDA-treated animals after intravenous primary immunization with SRBC. There are small but comparable numbers of PFC in the lymph node preparations from both the 6 - O H D A - and saline-treated animals suggesting that PFC are not migrating from the spleen to the lymph nodes. Changes in hemodynamics, antigen trapping and lymphocyte trafficking are still possible in the 6-OHDA-treated animals and could in part explain the results obtained. A trivial explanation for the effects noted is that 6 - O H D A is escaping from the brain into the periphery and thereby inducing 'direct' immunosuppression. When 100 ~g of 6 - O H D A was injected subcutaneously into animals followed by immunization 48 h later no effect on the primary SRBC antibody response was observed. Several additional observations as to the effects of 6 - O H D A on immune function warrant emphasis. When 6OHDA-treated animals are immunized with the thymus-independent antigen trinitrophenyl-lipopolysaccharide (TNP-LPS) no effect on this antibody response is noted. This suggests that the effects of 6 - O H D A are directed against the T-cell. Preliminary data indicate that 6 - O H D A treatment induces the appearance of T-suppressor cells (Ts). Treatment of animals with 6 - O H D A alone is not sufficient to induce Ts but rather requires that these animals must be treated both with 6 - O H D A and immunized with SRBC to induce the appearance of Ts. It remains to be established if these Ts are antigen-specific and these experiments are currently in progress. While these data describe the phenomenological effects of intracisternal injection of 6 - O H D A on the antibody response, both the neural and immunological mechanisms responsible for this effect are unknown. Moreover, it is tempting to ascribe immunoregulatory function solely to N E in this experimental model. Recent revidence that classical transmitters and regulatory neuropeptides can coexist in and be released from the same neuron indicates that these conclusions must be interpreted

65 with caution. Thus, catecholamine neurons also are capable to releasing enkephalin, neurotensin, neuropeptide Y, and cholecystokinin (reviewed in Hokfelt et al. 1984). Whereas these latter neuropeptides have no known effect on lymphocyte function, the enkephalins have been shown to modulate immune function (reviewed in Blalock 1984). Similar to the presumed mechanism(s) of endocrine-immune interaction, the possibility that neurotransmitters and/or neuropeptides may play a physiological role in the maintenance of immunocompetence is supported by the presence of receptors for certain of these transmitters on the surface membrane of lymphocytes, e.g., acetylcholine (Shapiro and Strom 1980), substance P (Payan et al. 1984), opiates and endorphin (Hazum et al. 1979). Beta-adrenergic receptors which are found on Tand B-cells also are linked to lymphocyte activation and are directly related to sympathetic nervous system activity (Miles et al. 1984). Thus, these data clearly demonstrate that alterations in neurotransmitters and peptides are capable of influencing the lymphoid elements of the immune system. The mechanism(s) by which this occurs remains to be explored. It is possible that the interaction of neurotransmitters with the appropriate lymphocyte receptor influences the metabolic activity of the cell by regulating intracellular cAMP, hence affecting the early events of lymphocyte activation as observed following central NE depletion. In addition, central catecholamine depletion does transiently affect the secretion of prolactin, growth and luteinizing hormones all of which could alter lymphocyte activity (Fenske and Wuttke 1976; Willoughby and Day 1981). The nature of alterations initiated by 6-OHDA treatment makes this a complex model to employ in the study of NIM. If, however, in vivo modulation of immune functions by 6-OHDA treatment can be studied in vitro, there is every reason to believe that the mechanisms can be elucidated. Alternatively, it is equally possible that removing the lymphoid cells from the in vivo environment will result in these cells reverting to a normal state in the absence of CNS-modulating substances, e.g., neurotransmitters, hormones, etc. Serotonin and the Immune Response Recently, we have begun to study the role of serotonin in mediating NIM (Jackson et al. 1985). These investigations are based on the report of Eremina and Devoino (1973) and Devoino et al. (I975), demonstrating that the humoral response to bovine serum albumin is enhanced following electrolytic lesioning of serotonergic neurons in the midbrain raphe nucleus. Hence the questions were asked: What is the effect of serotonin on the immune response? Is this neurotransmitter a CNS mediator of immune modulation? In the initial set of experiments mice were inoculated with either 5-hydroxytryptophan (5-HTP) which increases serotonin synthesis or parachlorophenylalanine (PCPA) which prevents the biosynthesis of serotonin by inhibiting the rate-limiting enzyme tryptophan hydroxylase prior to primary immunization with SRBC. The results of these experiments indicate that serotonin has immunomodulatory properties; increases in levels following the administration of 5-HTP result in significant suppression of the primary antibody response while diminished levels induced by

66 PCPA augment the response. As predicted based on the above findings, the administration of serotonin to animals prior to immunization was found to be inhibitory to the generation of antibody-forming cells. Thus, serotonin is capable of modulating both the IgM and IgG primary antibody response to SRBC and may be a neural messenger for immune modulation. Having established that serotonin may modulate humoral responsiveness, we then addressed the question: Are these effects mediated from the CNS or does serotonin have a direct effect on lymphocyte function? In these studies mice were injected intracisternally with 5,7-dihydroxytryptamine (5,7-DHT) which is a neurotoxin destroying both serotonergic and catecholamine-containing neurons in the brain. Some animals were also injected with desmethylimipramine (DMI) which blocks the uptake of 5,7-DHT by catecholamine neurons thereby limiting the effects of the neurotoxin to only serotonergic neurons. The results of these studies showed that injection of 5,7-DHT into animals pretreated with DMI has no effect on the PFC response to SRBC. These data suggest that immunomodulatory effects of serotonin are not mediated through neural pathways in the brain, but rather through peripheral mechanisms directed toward the lymphocyte itself. As a corollary, it may be suggested that receptors for serotonin, similar to those for adrenergic substances, will be present on the surface of the lymphocyte (Williams et al. 1976; Pochet et al. 1979). Recent evidence in our laboratory has shown that there are no receptors for serotonin on the lymphocyte membrane, yet a high affinity (Km = 10 -v M) active uptake system is observed with macrophages. Further work is required to establish the functional capabilities of this uptake system and its role in effecting the modulatory changes induced by serotonin. To date our studies have employed in vivo techniques to investigate the immunomodulatory effects of serotonin. These investigations have demonstrated that serotonin inhibits humoral responsiveness through mechanism(s) yet to be determined. Although our studies confirm others (Bliznakov 1980) that corticosterone does not play a significant role in this regulatory system, the role of other hormones under the influence of serotonergic pathways, e.g., growth hormone, prolactin, thyrotropin-releasing hormone, gonadotropins remains to be determined. It is evident, however, that these effects are not mediated through CNS pathways but rather directed toward lymphocyte function presumably via cellular uptake systems. More recently, we have attempted to extend the demonstration of serotonin's inhibitory properties to in vitro conditions in order to evaluate its effect on cellular responsiveness as well as to facilitate the exploration of its mechanism of action (Roszman et al. 1985). Although serotonin can be shown to inhibit lymphocyte mitogen-induced proliferation, the amounts required ( ] 0 - 4 10 - 3 M) are not physiologic but rather pharmacologic. Identical results were obtained with antigen-specific in vitro systems such as the SRBC primary antibody response and the response of turkey gamma globulin-specific long-term T-cells. Moreover, these concentrations of serotonin also inhibited the ability of splenic antigen-presenting cells to present antigen. We have found a number of activities that serotonin does not affect including the ability of LPS-stimulated macrophages to produce interleukin-1, elevated cAMP levels in spleen cell suspensions, induce suppressor cells and overall cell viability.

67 This raises the question as to the functional role of serotonin in regulating the immune response. Clearly alterations in peripheral serotonin are associated with modulations in immune reactivity. What is less evident, however, is how this is effected. Does serotonin influence lymphocyte responsiveness and immunity directly or does serotonin act as an intermediary for the reported alterations in immune responsiveness following variations in serotonin levels? It is also possible that serotonin achieves a high concentration in a local microenvironment in lymphoid tissue. This could result from increased release of serotonin from mast cells which are being regulated by neurotransmitters emanating from nerve terminals present in lymphoid tissue (Williams et al. 1981) or a T-cell-derived factor (Askenase et al. 1982). These observations clearly indicate that the immunological, physiological role of serotonin (and other neurotransmitters) must be dissected from its pharmacologic effects in order for the concepts of NIM to be considered as biologically relevant.

Closing Remarks Within recent years physical and biological sciences have been confronted with the principles of uncertainty and complementarity. This is especially notable in the experimental design for investigations of NIM. Immune functions which fall under the aegis of NIM may not be detectable in vitro where the interconnections of the CNS and mediators of immunity are severed and hence no longer functional. Lymphocyte responsiveness which may be modulated in the 'normal' milieu of the CNS and soluble products under its control, e.g., neurotransmitters, hormones, may behave quite differently in culture which in experiments of NIM must be considered non-physiological and indeed aberrant. Therefore, one is confronted with the uncertainty as to the biological significance as well as the relevancy of in vitro studies of NIM. Simply stated: Can NIM be explicated utilizing current in vitro techniques? This is an extremely important question as many of the reports that neurotransmitters and/or neurohormones modulate lymphocyte function employ pharmacological not physiological concentrations of the reputed mediator of NIM and at once raise serious concerns. The uncertainties of the correlation of in vivo and in vitro demonstrations of NIM are further compounded by consideration of the complementarity of the immune, central and peripheral nervous and endocrine systems. If the data supporting the hypothesis of NIM are true, perturbations in any one of these systems will effect alterations (presumably recordable) in the others. The question arises whether the effected alterations are specific or merely associative and thus of little, if any, biological significance. How these seemingly isolated systems communicate is of great interest and forms the basis for further study of NIM. Although investigations of NIM are difficult not only to properly design but also interpret, there is evidence that lymphocytes and neural cells make extensive use and respond to similar soluble messages. Receptors found on the surface of lymphocytes for hormones (Arrenbrecht 1974; Harrison et al. 1979) and neurotransmitters (Hazum et al. 1979; Pochet et al. 1979; Payan et al. 1984) as well as changes in neural reactivity inducible by lymphokines (Besedovsky et al. 1985) are indicative of

68 p o s s i b l e a v e n u e s for m e d i a t i o n b e t w e e n the C N S , e n d o c r i n e , a n d i m m u n e systems. T h e s e p o s s i b i l i t i e s are f u r t h e r s u b s t a n t i a t e d by the f i n d i n g s that a s t r o c y t e s p r o d u c e an i n t e r l e u k i n - l - l i k e s u b s t a n c e ( F o n t a n a et al. 1982) w h i l e l y m p h o c y t e s c a n be i n d u c e d to m a k e A C T H - l i k e a n d e n d o r p h i n - l i k e s u b s t a n c e s ( B l a l o c k 1984). T h u s , N I M m a y b e e f f e c t e d via a v a r i e t y of ' c y t o k i n e s ' p r o d u c e d by the b r a i n in the f o r m of h o r m o n e s a n d / o r n e u r o t r a n s m i t t e r s a n d f r o m the i m m u n e s y s t e m in the f o r m o f i n t e r l e u k i n s . It is the e l u c i d a t i o n of h o w these ' b l a c k b o x e s ' , neural, e n d o c r i n e , a n d i m m u n o l o g i c a l , c o m m u n i c a t e a n d are i n t e r - d e p e n d e n t in a b i o l o g i c a l r e l e v a n t netw o r k that is the a i m of i n v e s t i g a t i o n s of N I M . W i t h the n u m b e r of a p p r o p r i a t e m o d e l s y s t e m s a v a i l a b l e for s t u d y of t h e s e i n t e r a c t i o n s , the n e x t several y e a r s s h o u l d see these q u e s t i o n s r e s o l v e d a n d n e u r a l m o d u l a t i o n of i m m u n i t y b e c o m e m o r e t h a n conceptual and phenomenological.

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