Continuous vs. intermittent vagal activation is associated with similar suppression of T-cell-dependent antibody response as well as reduced body weight

Continuous vs. intermittent vagal activation is associated with similar suppression of T-cell-dependent antibody response as well as reduced body weight | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Continuous vs. intermittent vagal activation is associated with similar suppression of T-cell-dependent antibody response as well as reduced body weight Michael Gerber, Ibrahim Mughrabi, Izumi Kurata-Sato, Ethan Paliwoda, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9633685/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The vagus nerve is a regulator of immunity. However vagal activity in disease states remains poorly understood. Signaling between the vagus nerve and immune mechanisms is bi-directional: immune changes drive sensory vagal activity, and motor vagal tone alters immune responses. To isolate key drivers of vagal and immune dysfunction in diseases, we modeled a state of elevated vagal activation by delivering chronic VNS. We previously found that chronic, intermittent VNS results in an impaired T-dependent antibody response. We sought to investigate the impact of different patterns of vagal activation on antibody production. We delivered VNS to model an intermittently elevated state of vagal activation (Twice Daily VNS) and a chronically elevated state of vagal activation (Continuous Burst VNS) for 14 days before and 14 days after immunization with an antigen. We found that Continuous Burst VNS is associated with remodeling of cellular splenic compartments, as well as weight loss; such changes were not seen in Twice Daily VNS. High-affinity antibody production is reduced with both Continuous Burst VNS, compared to no VNS, as was previously found with Twice Daily VNS. Total IgG antibody titers are reduced in Continuous Burst VNS, even prior to immunization which is likely an effect of weight loss. In conclusion, elevated vagal tone is shown to limit the antibody response with different patterns of activation, however whole nerve stimulation is associated with confounding factors that limits interpretation at higher doses. Vagus Nerve Stimulation Neuromodulation Neuroimmune Stimulation Schedule Adaptive Immunity Germinal Center Reaction Figures Figure 1 Figure 2 Figure 3 Background The nervous system regulates immune function [ 1 ], with the vagus nerve playing a key role [ 2 ]. Bioelectronic activation of the vagus nerve suppresses acute inflammation through an anti-inflammatory pathway [ 3 ] that involves the splenic nerve [ 4 ] activating acetylcholine-producing T cells [ 5 ], resulting in activation of the alpha-7 nicotinic receptor on macrophages [ 6 ]. Neural control of the humoral immune response has also been documented [ 7 ], with the vagus, splenic nerve and lymphocyte relay activating alpha-7 and alpha-9 nicotinic receptors, resulting in impaired germinal center formation [ 8 , 9 ]. In addition to being a regulator of immunity, the vagus nerve senses inflammatory mediators [ 10 , 11 ] and relays these signals through dedicated fibers [ 12 ] to higher brain centers, which are suggested to serve a role in inflammatory memory [ 13 ]. The sensation, integration and motor output organization for these functions are mediated by the inflammatory reflex [ 1 ]. The fact that immunity is under neural control has led us, and others, to speculate on the role of the vagus in chronic immune dysfunction [ 14 ]. For example, the vagus is activated during sepsis [ 10 , 11 , 15 – 17 ], and while the initial anti-inflammatory effect is thought to be beneficial to organ function, persistent vagal activation may contribute to long-term adaptive immune dysfunction documented in sepsis survivors [ 18 ]. However, it is unknown whether chronic activation of the motor vagus drives such immune dysfunction or if immune dysfunction acts on the sensory vagus to shape an abnormal autonomic tone [ 17 ]. The natural firing pattern of the vagus, especially of the subset of fibers responsible for controlling and sensing inflammation [ 12 , 13 ], has not been fully described during or after inflammatory disease [ 17 ]. Understanding the role of the nervous system, and more specifically of the vagus nerve, within immune dysfunction and unraveling neuro-immune interactions will be critical for targeted therapy of sepsis and other diseases with neuro-immune dysfunction. To understand the effects of different patterns of vagal activity on immune function, we compared immunization response in a model of chronic VNS [ 19 ], under two distinct VNS schedules: twice daily stimulation trains during awake hours (Twice Daily) and continuous, frequent, short bursts over the 24 hour cycle (Continuous Burst) (Fig. 1 A). Twice Daily stimulation was previously found to influence innate [ 19 ] and adaptive immunity [ 8 ], while Continuous Burst may be eliciting vagal activation more similar to what occurs in sepsis [ 17 ]. Methods Animals and immunization Male C57BL/6 were purchased from Charles River Laboratories and used at 8 to 15 weeks of age. All mice were housed under specific pathogen–free conditions. Immunization with NP-CGG and blood sampling were performed as previously described [ 8 ]. All protocols including VNS were approved by the Institutional Animal Care and Use Committee. Chronic Implant Surgery Implants were prepared and implanted as previously described [ 19 ]. The same procedure was followed for animal housing and delivery of VNS as in our prior report [ 8 ]. Vagus nerve stimulation schedule Mice were stimulated on one of three schedules (Fig. 1 , A): No stimulation, 10-second trains every 5 minutes (Continuous Burst), or 5-minute trains twice daily (Twice Daily). Stimulation trains were made up of 500-µs pulses delivered at 30Hz, with current intensity adjusted to achieve ~ 20–30% reduction of baseline heart rate. To monitor heart rate, ECG was recorded and reviewed daily to monitor the functionality of the stimulating electrode and adjust stimulation intensity to maintain consistent heart rate responses throughout the VNS period. The average heart rate drop was calculated daily, and mice that were unresponsive even to maximum stimulus intensity (1.6mA) were dropped out. This criterion was met by one mouse from the Continuous Burst group, and implant dysfunction was confirmed on autopsy at the end of the treatment period. Typically, implants would elicit adequate heart rate drops at low stimulus intensities early during treatment but would require more intensity towards the end of treatment to maintain similar responses, likely due to fibrosis around the implant (Fig. 1 , B-C). Mice were monitored daily to observe indicators of well-being, including food and water consumption and nest building. VNS treatment was started on day − 14 relative to immunization, which was on day 0 (Fig. 1 , B-C). On day 0 and day 7, blood was collected by tail-tip sampling under isoflurane anesthesia for serological assays. At the end of day 14, mice were euthanized, and spleen and blood were collected for analysis. Enzyme-linked immunosorbent assay Serum titers of NP-specific antibodies, total IgG, and IgM were measured by enzyme-linked immunosorbent assay as we previously described [ 8 ]. Flow cytometry Splenocyte counts were obtained with flow cytometry using the same procedure, reagents and antibodies as previously described [ 8 ]. Immunohistochemistry Fresh spleens were embedded in TissueTek OCT (Sakura), quickly frozen over liquid nitrogen and sectioned at 10µm on a cryostat (Leica). Sectioned tissue was first fixed on the slide with 4% paraformaldehyde for 15 minutes, then incubated in Triton-X for 30 minutes, then blocked with 5% donkey serum and 10% BSA. Primary antibody was applied overnight at 4 o C, then secondary was applied for two hours at room temperature. Primary antibodies and dilutions are listed in Table 1 . Table 1 Primary antibodies and dilutions used for staining IHC sections. Antibody Host Dilution Vendor/Catalog B220 Rat 1:200 Biolegend/103239 CD3e Armenian Hamster 1:100 Biolegend/100312 Analysis and Statistics For all figures except NP2 IgG, NP25 IgG, and total IgG antibody titers, data is representative of one cohort of animals that were treated simultaneously; for the NP2 IgG, NP25 IgG and total IgG antibody titers, an additional cohort of No Stimulation (n = 3) vs Continuous Burst (n = 3) was included due to data availability. The combined cohorts were not statistically significant when compared to each other. ANCOVA was used to model the regression line for antibody titers over the 2 week post-immunization period between treatment groups and post-hoc pairwise comparisons were used to compare slopes and y-intercepts. One-way ANOVA with post-hoc pairwise comparisons was used to compare spleen cytology and histology outcome measures between treatment groups. For body weight data, multiple paired t-tests with Tukey-Kramer correction were used to make pairwise comparisons between each timepoint and baseline within treatment groups. For IHC, T cell, B cell, and red pulp areas were calculated as a percentage of the total spleen area. All IHC measurements represent an average of four sections spaced 100µm apart. All calculations were done in MATLAB using the Statistics and Machine Learning toolbox. Results & Discussion A) Antibody titers at day 0, 7, and 14 for NP2 IgG, B) NP25 IgG, C) Total IgG, D) NP25 IgM, E) Total IgM for No Stimulation, Twice Daily and Continuous Burst animals. Linear regression of antibody titers is plotted per group and slope and y-intercept is shown in the tables below. Continuous Burst stimulation reduces high affinity IgG, low affinity IgG and total IgG compared to No Stimulation Mice were subjected to two VNS paradigms (Fig. 1 ). Compared to No Stimulation, the rising slope of high-affinity NP2 IgG antibody titers is significantly decreased in Continuous Burst stimulation animals, but not in Twice Daily stimulation animals (Fig. 2 , A). We previously reported a reduction in NP2 IgG antibody production in Twice Daily stimulation animals [ 8 ]. In our present study, we estimated that Continuous Burst stimulation would have a greater effect on antibody production; thus the study was not designed to detect the subtle decrease in Twice Daily animals, however there is an emerging trend with Twice Daily animals having a lower slope compared to No Stimulation, but higher than Continuous Burst. Similar to our prior report in Twice Daily animals, there was no effect of Continuous Burst on NP25 IgG titers (Fig. 2 , B), however there is an emerging trend in total IgG with Continuous Burst having a lower y-intercept, suggesting lower levels of total IgG prior to immunization (Fig. 2 , C). Total IgM and NP25 IgM are largely unchanged in Continuous Burst animals, similar to prior findings in Twice Daily animals (Fig. 2 , D-E) [ 8 ]. These results suggest that chronically elevated vagus activity (Continuous Burst VNS) has similar and likely greater effect on high-affinity antibody production as previously reported with intermittent activity (Twice Daily VNS) [ 8 ]. Uniquely, Continuous Burst also features reduced total IgG titers prior to immunization that was not seen with Twice Daily animals. Total IgG titers are maintained through a balance between antibody production and clearance. Production is mostly dependent on secretion from long-lived plasma cells that depend on a bone marrow niche for survival and from short-lived plasma cells residing in secondary lymphoid organs [ 20 ]. It is not known if changes in neural signaling associated with VNS can alter long- or short-lived plasma cell differentiation or survival; it is unlikely that VNS directly modulates plasma cell survival through efferent cholinergic signaling, as the vagus does not innervate the bone marrow or lymph nodes [ 21 ], however it may indirectly activate sympathetic reflex circuits via afferent fiber activation [ 22 ] In the bone barrow, NE signaling has been shown to suppress CXCL12 expression [ 23 ], thereby affecting plasma cell homing and differentiation [ 24 ]. In lymph nodes, NE signaling may work through the β2-receptor to increase antibody secretion in primed B cells [ 25 ], though the contribution of short-lived plasma cells to total IgG titers is small compared to long-lived plasma cells [ 26 ]. However, aside from the effects of changing autonomic signaling, VNS has indirect effects on metabolism associated with chronic schedules similar to our Continuous Burst parameters [ 27 – 31 ], which may also influence antibody titers as well [ 32 – 34 ]. We next sought to determine if these metabolic effects are present in Continuous Burst animals and how they may affect immune function. A) Body weight, in grams, of No Stimulation (top), Twice Daily (middle), and Continuous Burst-stimulated animals (bottom). Comparison of B) spleen weight, C) total spleen cells, D) total B cells, E) total germinal center B cells, and F) NP + GCB cells, G) total CD4 + T cells, H) total CD8 + T cells between stimulation groups. I) Comparison of average cellular composition of spleens between stimulation groups. J) Examples of stained spleen sections (top) and segmented ROIs (bottom) used to compare K) T cell area, L) B cell area, and M) red pulp area between stimulation groups. Continuous Burst stimulation reduces total body weight, spleen size and total cellularity, but spares CD8 + T cells, compared to Twice Daily stimulation and No Stimulation. Continuous Burst stimulation caused a greater and more sustained reduction in body weight compared to Twice Daily (Fig. 3 , A). Continuous Burst animals have a significantly smaller spleen weight and fewer total splenocyte counts compared to Twice Daily animals (Fig. 3 , B-C). Vagal activation is known to suppress appetite [ 30 ], reduce gut fat absorption [ 31 ], and reduce body weight particularly with stimulation schedules resembling our Continuous Burst parameters (Fig. 1 , A) [ 27 – 29 , 35 ]. Calorie restriction models similarly show weight loss alongside reduced spleen weight and lower splenocyte counts [ 36 ]. Notably, weight loss alone can reduce antibody production [ 32 , 33 ] in calorie restriction models over a similar timeline to that in our study, which is the likely explanation for our findings with total IgG (Fig. 2 , C). Although body weight changes likely play a major role in the reduction of spleen weight and splenocyte count [ 37 ], there may be other effects related to the direct effects of autonomic neurotransmission as well. Spleen shrinkage was similarly reported in a brainstem lesion model of the anterior and medial hypothalamus [ 38 ], which are tonic inhibitors of sympathetic outflow [ 39 , 40 ], and lesion models in anatomically adjacent nuclei cause increased splenic nerve activity [ 41 ]; this indicates that chronic activation of the splenic nerve, a downstream effect of VNS [ 4 ], can lead to spleen shrinkage. Additionally, chronic treatment with adrenergic β2 agonists and the aforementioned hypothalamic lesion model share the feature that harvested splenocytes are resistant to proliferation via concanavalin A [ 38 , 42 ], implicating that these chronic adrenergic activation models share a feature of reduced splenocyte proliferation capacity. These varied reports support the possibility that spleen size reduction is related to chronically elevated splenic nerve tone through the direct actions of norepinephrine; however, the confounding effect of weight loss in our model limits further study. We further characterized the spleens in stimulated animals by measuring absolute counts of splenic B and T cells. B cells were markedly reduced in Continuous Burst stimulation (Fig. 3 , D), which may support our findings of reduced total IgG (Fig. 2 , C) [ 43 ]. Total B cell counts are known to be reduced in calorie restriction models [ 36 ], but there is limited evidence that direct effects of vagal activation, such as cholinergic or adrenergic receptor activation [ 5 , 22 , 44 ], can reduce B cell proliferation, production, or change egress from the spleen, and no such models have demonstrated reduced total B cell count in lymphoid organs. In contrast, nicotinic receptors have been shown to promote B cell survival in the spleen and increase production in the bone marrow [ 45 , 46 ]. We previously found that the germinal center reaction was limited in Twice Daily stimulated animals, with lower counts of antigen-specific germinal center B cells, but not total germinal center B cells [ 8 ]. In this study, Twice Daily stimulated animals are trending the same findings from our prior study but do not reach significance; as previously stated, the present study was not designed to capture the more subtle changes expected in Twice Daily VNS. In contrast, Continuous Burst animals have reduced total germinal center B cells as well as the NP+ germinal center B cell subset (Fig. 3 , E-F); this is likely due to a reduced total B cell population (Fig. 3 , D) that can participate in germinal center formation [ 47 ]. T cells were sub-divided into CD4 + and CD8 + sub-groups; only CD4 + are significantly reduced, while CD8 + totals are comparable to both other groups (Fig. 3 , G-H). Adrenergic signaling has been shown to reduce proliferation of T cells in lymphoid organs [ 48 ], while antagonism of alpha-1 receptors has been shown to increase lymphopoiesis in the thymus while altering lineage commitment to favor CD4 + cells over CD8 + through a mechanism suspected to involve reduced negative selection [ 49 ]. This suggests our findings of increased CD8+/CD4 + T cell ratio in the spleen may be related to increased thymic negative selection under influence of elevated adrenergic tone caused by reflex sympathetic activation from VNS [ 22 , 44 ]. However, sympathetic signaling has also been shown to drive CD8 + T cell, but not CD4 + T cell, egress from the spleen in the context of a 3 day treatment with angiotensin II [ 50 ]; our opposing findings suggest evolving dynamics of egress of lymphocytes in the progression of acute to chronic activation, or an indirect effect of other vagal circuits unrelated to the spleen. Weight loss is also a driving factor on the splenic T cell population, as calorie restriction models show increased apoptosis in both CD4 and CD8 subsets [ 51 ]. IHC area measurements of B-cell, T-cell and red pulp zones support our conclusions of a reduction in the size of all 3 compartments (Fig. 3 , I-J). Furthermore, the size reduction between B cell, T cell and red pulp was proportional (Fig. 3 , K-M). Norepinephrine has been shown to cause splenic contracture by stimulating smooth muscle in the splenic capsule [ 52 ] and to promote leukocyte egress from the spleen [ 53 , 54 ]; these mechanisms as well as the organ volume reduction seen with body weight loss [ 37 ] may explain these findings. The spleen being spared in Twice Daily VNS may be related to the lack of weight loss or to differences in splenic nerve activity expected between the two stimulation schedules [ 35 ]. These findings of spleen size and cellularity reduction alongside reduced antibody titers highlights the limitations of whole nerve VNS as a model for the study of neuroimmune interactions; the non-selective activation of circuits, such as those related to appetite and metabolism, can lead to confounding effects at higher doses such as weight loss, which limits the applicability of parametric studies at the upper limits of the physiologic norm. Conclusions In this brief report, we characterize a unique phenotype of continuously elevated vagal tone to better understand its effect on antibody production. We found that chronically elevated vagal activity results in reduced spleen size, splenocyte count and antibody titers that are not observed in intermittently elevated vagal activity; these outcomes are likely a result of direct and indirect effects of vagal fiber activation, such as weight loss. These results underscore a need for more fiber-specific vagal activation models to study the effect of vagal-spleen signaling on the neuroimmune niche within the spleen, as well as a potential area of interest for further study. Abbreviations VNS Vagus nerve stimulation NP CGG–4–hydroxy–3–nitrophenylacetyl chickenγ–globulin BSA Bovine serum albumin IHC Immunohistochemistry GCB Germinal center B cell TH Tyrosine Hydroxylase Declarations Ethics Approval and Consent to Participate All experiments were approved by the IACUC of the Feinstein Institute. Consent for Publication All authors consent to publication of this study. Competing Interests The authors declare no competing interests. Author’s Information Michael Gerber, Email: [email protected] Stavros Zanos, Email: [email protected] Funding This research was conducted using departmental funds from the Feinstein Institutes for Medical Research. Author Contribution MG, IM conceived, designed and performed experiments, analyzed data, interpreted data, and wrote the paper. IS performed experiments, analyzed data and interpreted data. EP performed experiments, analyzed data and wrote the paper. TAV performed experiments and analyzed data. BD and SZ conceived and designed experiments, analyzed data, interpreted data and wrote the paper. All authors approved the final version of this paper. Acknowledgement The authors thank Yong-Rui Zou, Valentin Pavlov and Sun Jung Kim for offering their expert advice for data interpretation and experimental planning. Data Availability All data generated or analyzed during this study are included in this published article. References Pavlov VA, Chavan SS, Tracey KJ. Molecular and Functional Neuroscience in Immunity. Annu Rev Immunol. 2018;36:783–812. Wu YJ, Wang L, Ji CF, Gu SF, Yin Q, Zuo J. The Role of alpha7nAChR-Mediated Cholinergic Anti-inflammatory Pathway in Immune Cells. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9633685","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":639699303,"identity":"3f07b602-d171-4b3b-926b-2fd51199211f","order_by":0,"name":"Michael Gerber","email":"","orcid":"","institution":"Donald and Barbara Zucker School of Medicine at Hofstra/Northwell","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Gerber","suffix":""},{"id":639699304,"identity":"1498ed5b-e344-4beb-a16d-249b3646c92c","order_by":1,"name":"Ibrahim Mughrabi","email":"","orcid":"","institution":"Feinstein Institutes for Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"","lastName":"Mughrabi","suffix":""},{"id":639699305,"identity":"6753f02d-b9c7-4663-9f4d-5f56523c7b49","order_by":2,"name":"Izumi Kurata-Sato","email":"","orcid":"","institution":"Feinstein Institutes for Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Izumi","middleName":"","lastName":"Kurata-Sato","suffix":""},{"id":639699306,"identity":"2790b985-7f44-496a-9249-6fb8998d8f60","order_by":3,"name":"Ethan Paliwoda","email":"","orcid":"","institution":"Albany Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ethan","middleName":"","lastName":"Paliwoda","suffix":""},{"id":639699307,"identity":"53606878-8860-4ce7-b499-c5b4731a9c6f","order_by":4,"name":"The Anh Vu","email":"","orcid":"","institution":"Elmezzi Graduate School of Molecular Medicine at Hofstra/Northwell","correspondingAuthor":false,"prefix":"","firstName":"The","middleName":"Anh","lastName":"Vu","suffix":""},{"id":639699309,"identity":"ef9a6fc0-d8c0-4c3c-a6bf-ae379b4a6b87","order_by":5,"name":"Betty Diamond","email":"","orcid":"","institution":"Donald and Barbara Zucker School of Medicine at Hofstra/Northwell","correspondingAuthor":false,"prefix":"","firstName":"Betty","middleName":"","lastName":"Diamond","suffix":""},{"id":639699311,"identity":"6741f4a1-43f0-4a0c-80c1-11e7dd7ae573","order_by":6,"name":"Stavros Zanos","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYPACGxDBBsQWMkDCgBgtaUDMDNIiwUOslsMkaJHvP/zswYc/5/P4pc8fe8xTAdTC3rxNAp8WgwPHzA1ntt0uluxLZjfmOQPUwnOsDL8WxgYzad6G24kbzjCzSee2AbVI5Jjh1SLfzP5NmufPOaiWf0At8m/wa2E4xmMmzcN2AKqlAWQLD34tBmd4yiRntiUnzuxhNpP+c0yCh40nrdgCr8P6j2+T+PDHLrGfh/GZ5IwaGzl+9sMbb+B1GAZgI035KBgFo2AUjAJsAAA/kTxYZJwW6QAAAABJRU5ErkJggg==","orcid":"","institution":"Donald and Barbara Zucker School of Medicine at Hofstra/Northwell","correspondingAuthor":true,"prefix":"","firstName":"Stavros","middleName":"","lastName":"Zanos","suffix":""}],"badges":[],"createdAt":"2026-05-06 17:08:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9633685/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9633685/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109760350,"identity":"dfc1a4e1-16cb-4981-b22f-75bd9923454d","added_by":"auto","created_at":"2026-05-22 07:28:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1716721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMice were subjected to 3 chronic VNS schedules: No Stimulation, Twice Daily and Continuous Burst, for 14 days before and 14 days after immunization with NP-CGG.\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eA) VNS stimulation schedules were administered daily to three groups of mice. B) Average heart rate drop and stimulus intensity over the 4-week treatment period from one representative animal in the Twice Daily group. Heart rate drop represents an average of 2 drops post-VNS per day. C) Same as B, but from the Continuous Burst group. Heart rate drop represents an average of 144 drops post-VNS per day.\u003c/p\u003e","description":"","filename":"Fig1FinalTiff.png","url":"https://assets-eu.researchsquare.com/files/rs-9633685/v1/03ab0b286bebf2477741f6e8.png"},{"id":109480692,"identity":"e276c0cb-53d9-4286-a6c2-518b3655e2bf","added_by":"auto","created_at":"2026-05-18 15:01:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2516125,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eContinuous burst stimulation reduces antibody production against NP-CGG.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Antibody titers at day 0, 7, and 14 for NP2 IgG, B) NP25 IgG, C) Total IgG, D) NP25 IgM, E) Total IgM for No Stimulation, Twice Daily and Continuous Burst animals. Linear regression of antibody titers is plotted per group and slope and y-intercept is shown in the tables below.\u003c/p\u003e","description":"","filename":"Fig2FinalTiff.png","url":"https://assets-eu.researchsquare.com/files/rs-9633685/v1/8ca0f8c4b742f11df5b2f0ad.png"},{"id":109759675,"identity":"ff88cbe7-a039-49b8-8eed-09dcc627736f","added_by":"auto","created_at":"2026-05-22 07:27:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12955943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eContinuous burst stimulation reduces body weight, spleen size and cellularity, but spares CD8+ T cells, compared to Twice Daily stimulation and No Stimulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Body weight, in grams, of No Stimulation (top), Twice Daily (middle), and Continuous Burst-stimulated animals (bottom). Comparison of B) spleen weight, C) total spleen cells, D) total B cells, E) total germinal center B cells, and F) NP+ GCB cells, G) total CD4+ T cells, H) total CD8+ T cells between stimulation groups. I) Comparison of average cellular composition of spleens between stimulation groups. J) Examples of stained spleen sections (top) and segmented ROIs (bottom) used to compare K) T cell area, L) B cell area, and M) red pulp area between stimulation groups.\u003c/p\u003e","description":"","filename":"Fig3FinalTiff.png","url":"https://assets-eu.researchsquare.com/files/rs-9633685/v1/c7b87a835e7f141231d2b2fb.png"},{"id":109911853,"identity":"a8cf3363-b4bf-48ab-b889-cb715181483e","added_by":"auto","created_at":"2026-05-25 07:25:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17491751,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9633685/v1/beb1b10b-ea5b-4f73-821a-22a83dda3767.pdf"},{"id":109480691,"identity":"7ffe7917-a297-4d1c-bf2d-fe806e16fcfe","added_by":"auto","created_at":"2026-05-18 15:01:48","extension":"tiff","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14061542,"visible":true,"origin":"","legend":"","description":"","filename":"Table1FinalTiff.tiff","url":"https://assets-eu.researchsquare.com/files/rs-9633685/v1/c37486a3b7dae10ba4bbc578.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Continuous vs. intermittent vagal activation is associated with similar suppression of T-cell-dependent antibody response as well as reduced body weight","fulltext":[{"header":"Background","content":"\u003cp\u003eThe nervous system regulates immune function [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], with the vagus nerve playing a key role [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Bioelectronic activation of the vagus nerve suppresses acute inflammation through an anti-inflammatory pathway [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] that involves the splenic nerve [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] activating acetylcholine-producing T cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], resulting in activation of the alpha-7 nicotinic receptor on macrophages [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Neural control of the humoral immune response has also been documented [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], with the vagus, splenic nerve and lymphocyte relay activating alpha-7 and alpha-9 nicotinic receptors, resulting in impaired germinal center formation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition to being a regulator of immunity, the vagus nerve senses inflammatory mediators [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and relays these signals through dedicated fibers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] to higher brain centers, which are suggested to serve a role in inflammatory memory [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The sensation, integration and motor output organization for these functions are mediated by the inflammatory reflex [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe fact that immunity is under neural control has led us, and others, to speculate on the role of the vagus in chronic immune dysfunction [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For example, the vagus is activated during sepsis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and while the initial anti-inflammatory effect is thought to be beneficial to organ function, persistent vagal activation may contribute to long-term adaptive immune dysfunction documented in sepsis survivors [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, it is unknown whether chronic activation of the motor vagus drives such immune dysfunction or if immune dysfunction acts on the sensory vagus to shape an abnormal autonomic tone [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The natural firing pattern of the vagus, especially of the subset of fibers responsible for controlling and sensing inflammation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], has not been fully described during or after inflammatory disease [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Understanding the role of the nervous system, and more specifically of the vagus nerve, within immune dysfunction and unraveling neuro-immune interactions will be critical for targeted therapy of sepsis and other diseases with neuro-immune dysfunction.\u003c/p\u003e \u003cp\u003eTo understand the effects of different patterns of vagal activity on immune function, we compared immunization response in a model of chronic VNS [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], under two distinct VNS schedules: twice daily stimulation trains during awake hours (Twice Daily) and continuous, frequent, short bursts over the 24 hour cycle (Continuous Burst) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Twice Daily stimulation was previously found to influence innate [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and adaptive immunity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], while Continuous Burst may be eliciting vagal activation more similar to what occurs in sepsis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and immunization\u003c/h2\u003e \u003cp\u003eMale C57BL/6 were purchased from Charles River Laboratories and used at 8 to 15 weeks of age. All mice were housed under specific pathogen\u0026ndash;free conditions. Immunization with NP-CGG and blood sampling were performed as previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. All protocols including VNS were approved by the Institutional Animal Care and Use Committee.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChronic Implant Surgery\u003c/h3\u003e\n\u003cp\u003eImplants were prepared and implanted as previously described [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The same procedure was followed for animal housing and delivery of VNS as in our prior report [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eVagus nerve stimulation schedule\u003c/h3\u003e\n\u003cp\u003eMice were stimulated on one of three schedules (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, A): No stimulation, 10-second trains every 5 minutes (Continuous Burst), or 5-minute trains twice daily (Twice Daily). Stimulation trains were made up of 500-\u0026micro;s pulses delivered at 30Hz, with current intensity adjusted to achieve\u0026thinsp;~\u0026thinsp;20\u0026ndash;30% reduction of baseline heart rate. To monitor heart rate, ECG was recorded and reviewed daily to monitor the functionality of the stimulating electrode and adjust stimulation intensity to maintain consistent heart rate responses throughout the VNS period. The average heart rate drop was calculated daily, and mice that were unresponsive even to maximum stimulus intensity (1.6mA) were dropped out. This criterion was met by one mouse from the Continuous Burst group, and implant dysfunction was confirmed on autopsy at the end of the treatment period. Typically, implants would elicit adequate heart rate drops at low stimulus intensities early during treatment but would require more intensity towards the end of treatment to maintain similar responses, likely due to fibrosis around the implant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, B-C). Mice were monitored daily to observe indicators of well-being, including food and water consumption and nest building. VNS treatment was started on day\u0026thinsp;\u0026minus;\u0026thinsp;14 relative to immunization, which was on day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, B-C). On day 0 and day 7, blood was collected by tail-tip sampling under isoflurane anesthesia for serological assays. At the end of day 14, mice were euthanized, and spleen and blood were collected for analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEnzyme-linked immunosorbent assay\u003c/h3\u003e\n\u003cp\u003eSerum titers of NP-specific antibodies, total IgG, and IgM were measured by enzyme-linked immunosorbent assay as we previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003eSplenocyte counts were obtained with flow cytometry using the same procedure, reagents and antibodies as previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eFresh spleens were embedded in TissueTek OCT (Sakura), quickly frozen over liquid nitrogen and sectioned at 10\u0026micro;m on a cryostat (Leica). Sectioned tissue was first fixed on the slide with 4% paraformaldehyde for 15 minutes, then incubated in Triton-X for 30 minutes, then blocked with 5% donkey serum and 10% BSA. Primary antibody was applied overnight at 4\u003csup\u003eo\u003c/sup\u003eC, then secondary was applied for two hours at room temperature. Primary antibodies and dilutions are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimary antibodies and dilutions used for staining IHC sections.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHost\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVendor/Catalog\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend/103239\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD3e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArmenian Hamster\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiolegend/100312\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnalysis and Statistics\u003c/h3\u003e\n\u003cp\u003eFor all figures except NP2 IgG, NP25 IgG, and total IgG antibody titers, data is representative of one cohort of animals that were treated simultaneously; for the NP2 IgG, NP25 IgG and total IgG antibody titers, an additional cohort of No Stimulation (n\u0026thinsp;=\u0026thinsp;3) vs Continuous Burst (n\u0026thinsp;=\u0026thinsp;3) was included due to data availability. The combined cohorts were not statistically significant when compared to each other. ANCOVA was used to model the regression line for antibody titers over the 2 week post-immunization period between treatment groups and post-hoc pairwise comparisons were used to compare slopes and y-intercepts. One-way ANOVA with post-hoc pairwise comparisons was used to compare spleen cytology and histology outcome measures between treatment groups. For body weight data, multiple paired t-tests with Tukey-Kramer correction were used to make pairwise comparisons between each timepoint and baseline within treatment groups. For IHC, T cell, B cell, and red pulp areas were calculated as a percentage of the total spleen area. All IHC measurements represent an average of four sections spaced 100\u0026micro;m apart. All calculations were done in MATLAB using the Statistics and Machine Learning toolbox.\u003c/p\u003e"},{"header":"Results \u0026 Discussion","content":"\u003cp\u003e \u003c/p\u003e \u003cp\u003eA) Antibody titers at day 0, 7, and 14 for NP2 IgG, B) NP25 IgG, C) Total IgG, D) NP25 IgM, E) Total IgM for No Stimulation, Twice Daily and Continuous Burst animals. Linear regression of antibody titers is plotted per group and slope and y-intercept is shown in the tables below.\u003c/p\u003e \u003cp\u003e \u003cb\u003eContinuous Burst stimulation reduces high affinity IgG, low affinity IgG and total IgG compared to No Stimulation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMice were subjected to two VNS paradigms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compared to No Stimulation, the rising slope of high-affinity NP2 IgG antibody titers is significantly decreased in Continuous Burst stimulation animals, but not in Twice Daily stimulation animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, A). We previously reported a reduction in NP2 IgG antibody production in Twice Daily stimulation animals [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In our present study, we estimated that Continuous Burst stimulation would have a greater effect on antibody production; thus the study was not designed to detect the subtle decrease in Twice Daily animals, however there is an emerging trend with Twice Daily animals having a lower slope compared to No Stimulation, but higher than Continuous Burst. Similar to our prior report in Twice Daily animals, there was no effect of Continuous Burst on NP25 IgG titers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, B), however there is an emerging trend in total IgG with Continuous Burst having a lower y-intercept, suggesting lower levels of total IgG prior to immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, C). Total IgM and NP25 IgM are largely unchanged in Continuous Burst animals, similar to prior findings in Twice Daily animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, D-E) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese results suggest that chronically elevated vagus activity (Continuous Burst VNS) has similar and likely greater effect on high-affinity antibody production as previously reported with intermittent activity (Twice Daily VNS) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Uniquely, Continuous Burst also features reduced total IgG titers prior to immunization that was not seen with Twice Daily animals. Total IgG titers are maintained through a balance between antibody production and clearance. Production is mostly dependent on secretion from long-lived plasma cells that depend on a bone marrow niche for survival and from short-lived plasma cells residing in secondary lymphoid organs [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It is not known if changes in neural signaling associated with VNS can alter long- or short-lived plasma cell differentiation or survival; it is unlikely that VNS directly modulates plasma cell survival through efferent cholinergic signaling, as the vagus does not innervate the bone marrow or lymph nodes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], however it may indirectly activate sympathetic reflex circuits via afferent fiber activation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] In the bone barrow, NE signaling has been shown to suppress CXCL12 expression [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], thereby affecting plasma cell homing and differentiation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In lymph nodes, NE signaling may work through the β2-receptor to increase antibody secretion in primed B cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], though the contribution of short-lived plasma cells to total IgG titers is small compared to long-lived plasma cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, aside from the effects of changing autonomic signaling, VNS has indirect effects on metabolism associated with chronic schedules similar to our Continuous Burst parameters [\u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which may also influence antibody titers as well [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. We next sought to determine if these metabolic effects are present in Continuous Burst animals and how they may affect immune function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA) Body weight, in grams, of No Stimulation (top), Twice Daily (middle), and Continuous Burst-stimulated animals (bottom). Comparison of B) spleen weight, C) total spleen cells, D) total B cells, E) total germinal center B cells, and F) NP\u0026thinsp;+\u0026thinsp;GCB cells, G) total CD4\u0026thinsp;+\u0026thinsp;T cells, H) total CD8\u0026thinsp;+\u0026thinsp;T cells between stimulation groups. I) Comparison of average cellular composition of spleens between stimulation groups. J) Examples of stained spleen sections (top) and segmented ROIs (bottom) used to compare K) T cell area, L) B cell area, and M) red pulp area between stimulation groups.\u003c/p\u003e \u003cp\u003e \u003cb\u003eContinuous Burst stimulation reduces total body weight, spleen size and total cellularity, but spares CD8\u0026thinsp;+\u0026thinsp;T cells, compared to Twice Daily stimulation and No Stimulation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eContinuous Burst stimulation caused a greater and more sustained reduction in body weight compared to Twice Daily (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, A). Continuous Burst animals have a significantly smaller spleen weight and fewer total splenocyte counts compared to Twice Daily animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, B-C). Vagal activation is known to suppress appetite [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], reduce gut fat absorption [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and reduce body weight particularly with stimulation schedules resembling our Continuous Burst parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, A) [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Calorie restriction models similarly show weight loss alongside reduced spleen weight and lower splenocyte counts [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Notably, weight loss alone can reduce antibody production [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] in calorie restriction models over a similar timeline to that in our study, which is the likely explanation for our findings with total IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, C).\u003c/p\u003e \u003cp\u003eAlthough body weight changes likely play a major role in the reduction of spleen weight and splenocyte count [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], there may be other effects related to the direct effects of autonomic neurotransmission as well. Spleen shrinkage was similarly reported in a brainstem lesion model of the anterior and medial hypothalamus [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], which are tonic inhibitors of sympathetic outflow [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and lesion models in anatomically adjacent nuclei cause increased splenic nerve activity [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]; this indicates that chronic activation of the splenic nerve, a downstream effect of VNS [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], can lead to spleen shrinkage. Additionally, chronic treatment with adrenergic β2 agonists and the aforementioned hypothalamic lesion model share the feature that harvested splenocytes are resistant to proliferation via concanavalin A [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], implicating that these chronic adrenergic activation models share a feature of reduced splenocyte proliferation capacity. These varied reports support the possibility that spleen size reduction is related to chronically elevated splenic nerve tone through the direct actions of norepinephrine; however, the confounding effect of weight loss in our model limits further study.\u003c/p\u003e \u003cp\u003eWe further characterized the spleens in stimulated animals by measuring absolute counts of splenic B and T cells. B cells were markedly reduced in Continuous Burst stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, D), which may support our findings of reduced total IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, C) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Total B cell counts are known to be reduced in calorie restriction models [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], but there is limited evidence that direct effects of vagal activation, such as cholinergic or adrenergic receptor activation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], can reduce B cell proliferation, production, or change egress from the spleen, and no such models have demonstrated reduced total B cell count in lymphoid organs. In contrast, nicotinic receptors have been shown to promote B cell survival in the spleen and increase production in the bone marrow [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. We previously found that the germinal center reaction was limited in Twice Daily stimulated animals, with lower counts of antigen-specific germinal center B cells, but not total germinal center B cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In this study, Twice Daily stimulated animals are trending the same findings from our prior study but do not reach significance; as previously stated, the present study was not designed to capture the more subtle changes expected in Twice Daily VNS. In contrast, Continuous Burst animals have reduced total germinal center B cells as well as the NP+ germinal center B cell subset (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, E-F); this is likely due to a reduced total B cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, D) that can participate in germinal center formation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eT cells were sub-divided into CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;sub-groups; only CD4\u0026thinsp;+\u0026thinsp;are significantly reduced, while CD8\u0026thinsp;+\u0026thinsp;totals are comparable to both other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, G-H). Adrenergic signaling has been shown to reduce proliferation of T cells in lymphoid organs [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], while antagonism of alpha-1 receptors has been shown to increase lymphopoiesis in the thymus while altering lineage commitment to favor CD4\u0026thinsp;+\u0026thinsp;cells over CD8\u0026thinsp;+\u0026thinsp;through a mechanism suspected to involve reduced negative selection [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This suggests our findings of increased CD8+/CD4\u0026thinsp;+\u0026thinsp;T cell ratio in the spleen may be related to increased thymic negative selection under influence of elevated adrenergic tone caused by reflex sympathetic activation from VNS [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, sympathetic signaling has also been shown to drive CD8\u0026thinsp;+\u0026thinsp;T cell, but not CD4\u0026thinsp;+\u0026thinsp;T cell, egress from the spleen in the context of a 3 day treatment with angiotensin II [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]; our opposing findings suggest evolving dynamics of egress of lymphocytes in the progression of acute to chronic activation, or an indirect effect of other vagal circuits unrelated to the spleen. Weight loss is also a driving factor on the splenic T cell population, as calorie restriction models show increased apoptosis in both CD4 and CD8 subsets [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIHC area measurements of B-cell, T-cell and red pulp zones support our conclusions of a reduction in the size of all 3 compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, I-J). Furthermore, the size reduction between B cell, T cell and red pulp was proportional (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, K-M). Norepinephrine has been shown to cause splenic contracture by stimulating smooth muscle in the splenic capsule [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] and to promote leukocyte egress from the spleen [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]; these mechanisms as well as the organ volume reduction seen with body weight loss [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] may explain these findings. The spleen being spared in Twice Daily VNS may be related to the lack of weight loss or to differences in splenic nerve activity expected between the two stimulation schedules [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These findings of spleen size and cellularity reduction alongside reduced antibody titers highlights the limitations of whole nerve VNS as a model for the study of neuroimmune interactions; the non-selective activation of circuits, such as those related to appetite and metabolism, can lead to confounding effects at higher doses such as weight loss, which limits the applicability of parametric studies at the upper limits of the physiologic norm.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this brief report, we characterize a unique phenotype of continuously elevated vagal tone to better understand its effect on antibody production. We found that chronically elevated vagal activity results in reduced spleen size, splenocyte count and antibody titers that are not observed in intermittently elevated vagal activity; these outcomes are likely a result of direct and indirect effects of vagal fiber activation, such as weight loss. These results underscore a need for more fiber-specific vagal activation models to study the effect of vagal-spleen signaling on the neuroimmune niche within the spleen, as well as a potential area of interest for further study.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVNS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVagus nerve stimulation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCGG\u0026ndash;4\u0026ndash;hydroxy\u0026ndash;3\u0026ndash;nitrophenylacetyl chickenγ\u0026ndash;globulin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBovine serum albumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGCB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGerminal center B cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTyrosine Hydroxylase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e \u003cp\u003eAll experiments were approved by the IACUC of the Feinstein Institute.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for Publication\u003c/strong\u003e \u003cp\u003eAll authors consent to publication of this study.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthor\u0026rsquo;s Information\u003c/h2\u003e \u003cp\u003eMichael Gerber, Email: [email protected]\u003c/p\u003e \u003cp\u003eStavros Zanos, Email: [email protected]\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was conducted using departmental funds from the Feinstein Institutes for Medical Research.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMG, IM conceived, designed and performed experiments, analyzed data, interpreted data, and wrote the paper. IS performed experiments, analyzed data and interpreted data. EP performed experiments, analyzed data and wrote the paper. TAV performed experiments and analyzed data. BD and SZ conceived and designed experiments, analyzed data, interpreted data and wrote the paper. All authors approved the final version of this paper.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Yong-Rui Zou, Valentin Pavlov and Sun Jung Kim for offering their expert advice for data interpretation and experimental planning.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePavlov VA, Chavan SS, Tracey KJ. Molecular and Functional Neuroscience in Immunity. 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Exp Neurol. 2009;218(1):47\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioelectronic-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"beme","sideBox":"Learn more about [Bioelectronic Medicine](https://bioelecmed.biomedcentral.com)","snPcode":"42234","submissionUrl":"https://submission.springernature.com/new-submission/42234/3","title":"Bioelectronic Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Vagus Nerve Stimulation, Neuromodulation, Neuroimmune, Stimulation Schedule, Adaptive Immunity, Germinal Center Reaction","lastPublishedDoi":"10.21203/rs.3.rs-9633685/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9633685/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe vagus nerve is a regulator of immunity. However vagal activity in disease states remains poorly understood. Signaling between the vagus nerve and immune mechanisms is bi-directional: immune changes drive sensory vagal activity, and motor vagal tone alters immune responses. To isolate key drivers of vagal and immune dysfunction in diseases, we modeled a state of elevated vagal activation by delivering chronic VNS. We previously found that chronic, intermittent VNS results in an impaired T-dependent antibody response. We sought to investigate the impact of different patterns of vagal activation on antibody production. We delivered VNS to model an intermittently elevated state of vagal activation (Twice Daily VNS) and a chronically elevated state of vagal activation (Continuous Burst VNS) for 14 days before and 14 days after immunization with an antigen. We found that Continuous Burst VNS is associated with remodeling of cellular splenic compartments, as well as weight loss; such changes were not seen in Twice Daily VNS. High-affinity antibody production is reduced with both Continuous Burst VNS, compared to no VNS, as was previously found with Twice Daily VNS. Total IgG antibody titers are reduced in Continuous Burst VNS, even prior to immunization which is likely an effect of weight loss. In conclusion, elevated vagal tone is shown to limit the antibody response with different patterns of activation, however whole nerve stimulation is associated with confounding factors that limits interpretation at higher doses.\u003c/p\u003e","manuscriptTitle":"Continuous vs. intermittent vagal activation is associated with similar suppression of T-cell-dependent antibody response as well as reduced body weight","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 15:01:43","doi":"10.21203/rs.3.rs-9633685/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-25T07:09:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-18T18:23:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"19733905069979498195165099820757831124","date":"2026-05-13T09:13:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180205717147450783410238454085827975860","date":"2026-05-12T15:14:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321030459239722853093021808820418981515","date":"2026-05-11T07:34:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-07T19:57:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-07T02:07:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-07T02:07:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioelectronic Medicine","date":"2026-05-06T16:55:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioelectronic-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"beme","sideBox":"Learn more about [Bioelectronic Medicine](https://bioelecmed.biomedcentral.com)","snPcode":"42234","submissionUrl":"https://submission.springernature.com/new-submission/42234/3","title":"Bioelectronic Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2d3d7778-a4b6-46cc-9dcb-2a38ed2d51c9","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-25T07:09:15+00:00","index":29,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-18T18:23:41+00:00","index":28,"fulltext":""},{"type":"reviewerAgreed","content":"19733905069979498195165099820757831124","date":"2026-05-13T09:13:44+00:00","index":27,"fulltext":""},{"type":"reviewerAgreed","content":"180205717147450783410238454085827975860","date":"2026-05-12T15:14:36+00:00","index":26,"fulltext":""},{"type":"reviewerAgreed","content":"321030459239722853093021808820418981515","date":"2026-05-11T07:34:18+00:00","index":17,"fulltext":""},{"type":"reviewersInvited","content":"13","date":"2026-05-07T19:57:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-07T02:07:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-07T02:07:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioelectronic Medicine","date":"2026-05-06T16:55:39+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T15:01:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 15:01:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9633685","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9633685","identity":"rs-9633685","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}