Neuronal Circuits Modulate Antigen Flow Through Lymph Nodes
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When pathogens and toxins breech the epithelial barrier, antigens are transported by the lymphatic system to lymph nodes. In previously immunized animals, antigens become trapped in the draining lymph nodes, but the underlying mechanism that controls antigen restriction is poorly understood. Here we describe the role of neurons in sensing and restricting antigen flow in lymph nodes. The antigen keyhole-limpet hemocyanin (KLH) injected into the mouse hind paw flows from the popliteal lymph node to the sciatic lymph node, continuing through the upper lymphatics to reach the systemic circulation. Re-exposure to KLH in previously immunized mice leads to decreased flow from the popliteal to the sciatic lymph node as compared with naïve mice. Administering bupivacaine into the lymph node region restores antigen flow in immunized animals. In contrast, neural activation using magnetic stimulation significantly decreases antigen trafficking in naïve animals as compared with sham controls. Ablating NaV1.8 + sensory neurons significantly reduces antigen restriction in immunized mice. Genetic deletion of FcγRI/FcεRI also reverses the antigen restriction. Colocalization of PGP9.5-expressing neurons, FcγRI receptors and labeled antigen occurs at the antigen challenge site. Together, these studies reveal that neuronal circuits modulate antigen trafficking through a pathway that requires NaV1.8 and FcγR.
The immune and nervous systems coevolved under the threat of environmental dangers and work in concert to sense changes in the environment, mobilize host responses and establish memories of threatening events. The nervous system communicates with the immune system via direct innervation of primary and secondary lymphoid organs. These lymphoid organs play an essential role in peripheral inflammatory responses to immunological challenge and/or tissue injury. Antigens are transported to the local lymph nodes, processed by antigen-presenting cells and presented to lymphocytes to trigger an immune response. Antigen-specific T and B cells are primed and activated in the draining lymph nodes, resulting in antigen-specific acute and memory responses. Controlling the lymphatic transit of antigens to these draining lymph nodes is essential to mount an efficient host defense against pathogens, and is subject to several regulatory mechanisms, including the nervous system.
A dense plexus of neural fibers is present in blood vessels and the surrounding periarterial lymphatic sheath in lymph nodes (1). Lymph nodes are also richly innervated by both afferent and efferent nerve fibers. Detailed microscopy studies have revealed abundant innervation of the lymph node parenchyma, with specific innervation targeting single antigen-presenting cells (2). Cross-talk between local neural and immune responses modulates inflammatory and neural input to lymph nodes. Antigenic stimulation leads to an increase in the density of lymph node innervation (3). In a contact sensitivity model, antigenic challenge induces an increase in the length of nerve fibers (4). Subcutaneous injection of antigen is associated with increased nerve-ending density in the medulla of the draining lymph node, indicating functional activation of the autonomic nervous system through the immune system (5). Catecholamine released during acute psychological stress leads to mobilization of lymphocytes into the blood (6). Direct neural stimulation leads to increased output of lymphocytes from the popliteal lymph nodes (7). In contrast, silencing local neural input reduces lymphocyte output from lymph nodes by more than 99%, indicating that neural signals control leukocyte retention and trafficking through the lymph nodes (8).
Recent studies have demonstrated that sensory nociceptor neurons that express the ion channels TRPV1 and NaV1.8 are activated directly by bacteria, leading to modulation of pain and inflammation (9). These nociceptors are also essential for developing allergic inflammatory responses in allergic conditions and cutaneous inflammation (10, 11, 12). Nociceptors have been implicated in modulating lymph node function, because ablation of nociceptor neurons attenuates hypertrophy in draining lymph nodes and skin inflammatory responses in psoriasis (12). Specific ablation of NaV1.8-lineage nociceptor neurons abrogates pain during bacterial infection, but concurrently increases local immune infiltration and lymphadenopathy in the draining lymph nodes (9). Together these observations indicate that nociceptor neurons are activated by exogenous stimuli and modulate lymph node trafficking. However, the specific subset of peripheral neurons that control the lymphatic transit has not yet been characterized. Here, we investigated the transit of a labeled antigen through the lymphatic system and identified the TRPV1−/NaV1.8+ population of peripheral neurons as required for neuronal input leading to antigen restriction.
Materials and Methods
Keyhole limpet hemocyanin (KLH, Calbiochem) and ovalbumin (OVA, Sigma-Aldrich) were diluted to 1 mg/mL with phosphate-buffered saline (PBS), then potassium phosphate dibasic, pH 9.0, was added to 0.1M final concentration. Then 0.5 mg IrDye (800CW-NHS ester or 680LT-NHS ester, Licor) or Alexa Fluor® 647 NHS Ester (A647, Thermo Scientific) was added for each 25 mg protein; 1 mL aliquots were then incubated at 20°C with 300 RPM, shaking for 2 h. Labelled antigen was separated by centrifugation through PBS-washed Zeba spin desalting columns (Thermo Scientific). Dye-labeled antigens were concentrated with 100K (KLH) or 30K MWCO (OVA) spin columns (Millipore).
Labeled antigens were loaded on NuPAGE 4–12% Bis-Tris Gels (Invitrogen) with NuPAGE MOPS SDS Running Buffer and run according to the manufacturer’s instructions, 50 min at 200 constant volts. Gels were extracted from cassettes and imaged directly on an Odyssey infrared imager (Licor).
Balb/c, B6.129P2-Gt(ROSA)26Sortm1(DTA)Lky (floxed-stop-DTA), B6.129-Trpv1tm1(cre)Bbm (TRPV1-cre) and B6;129P2-Fcer1gtm1Rav (FcR KO) mice were obtained from Jackson Laboratories. FcR KO mice lack the Fcγ adaptor protein, which transduces FcγRI and FcεRI signaling. NaV1.8-Cre+/− mice (13) were a gift to CW from R. Kuner (University of Heidelberg). NaV1.8-Cre+/− mice were bred with C57BL/6 DTA+/+ mice to generate nociceptor-deficient NaV1.8-Cre+/−/DTA+/− and control littermates (NaV1.8-Cre−/−/DTA+/−). TRPV1-Cre+/− mice were bred with C57BL/6 DTA+/+ mice to generate TRPV1-Cre+/−/DTA+/− and control littermates (TRPV1-Cre−/−/DTA+/−). Food and water were available ad libitum. Mice were used in subsequent experiments after at least a 14 d adaptation period. All procedures were performed in accordance with National Institutes of Health Guidelines (14) under protocols approved by the Institutional Animal Care and Use Committee of The Feinstein Institute for Medical Research.
Mice were immunized with 100 µg antigen and 50% Imject alum in 200 µl saline, injected intraperitoneally twice, 2 wks apart. Two weeks after the second injection, mice were injected with KLH-A647 in the dorsum of the hind paw. After 1 h, skin around the injection site was excised, frozen in optimal cutting temperature media (Tissue-Tek), sliced at 10 µm and mounted on Superfrost/Plus slides (Fisher Scientific). Sections were blocked with 5% goat serum and rat anti-CD16/CD32 (BD Biosciences). Sections were then stained for 1 h with rabbit anti-PGP9.5 (EMD Millipore) and mouse anti-CD64 (Biolegend, clone × 54–5/7.1). Secondary antibodies were goat anti-mouse IgG-Dylight 550 and goat anti-rabbit IgG-Dylight 488 (Thermo Scientific). Images were obtained on a FluoView FV300 laser-scanning confocal microscope (Olympus).
Unlabeled antigen (100 µg) and 50% Imject alum (Thermo Scientific) in 200 µl of 0.9% saline was injected intraperitoneally. Two weeks later, animals were injected again with the same antigen emulsion as a booster. Two weeks after the booster injection, animals were studied in experiments. Control animals received injections of 50% alum in saline. For passive immunization, naïve Balb/c mice were injected intraperitoneally with 100 µg rabbit polyclonal anti-ovalbumin antibody (Millipore). After 24 h, mice were injected with OVA-800CW or KLH-800CW subcutaneously in the dorsum of the hind paw.
In Situ Antigen Imaging
Using an insulin syringe, 20 µl of 100 µg labeled antigen was injected subcutaneously into the dorsum of the hind paw. At the end of the experiment, the animal was euthanized using carbon dioxide asphyxiation, the skin over the area of interest was removed and the animal was placed in the supine position in an Odyssey infrared imager to detect the fluorescence of labeled antigen in the popliteal and sciatic lymph nodes. Fluorescent antigen signal was expressed as arbitrary units (A.U.). To image lymph node antigen content, sciatic and popliteal lymph nodes were surgically removed and placed into black, clear-bottomed 96-well plates filled with T-per (Thermo Scientific). These plates were imaged on the Odyssey imager.
Antigen-Specific Antibody Titer
High-binding 96-well microplates (Corning) were coated using 20 µg/mL KLH in PBS and incubated overnight at room temperature. The following day, the plates were washed with PBS + 0.01% Tween20 and blocked with 1 mg/mL bovine serum albumin in PBS. Serum from immunized animals was obtained by cardiac puncture followed by centrifugation at 2,000 × g for 10 min. Serum was diluted 1:100–1:10,000 with PBS and 100 µl per sample was added to washed and blocked plates. Plates were incubated for 2 h, followed by incubation with 1:2000 sheep-derived anti-mouse IgM-HRP (BD) or anti-mouse IgG (Amersham) for 2 h. Plates were washed a final time, developed using Opt-EIA (BD) and the reaction was stopped with H2SO4.
Mice were immunized with 100 µg antigen and 50% Imject alum in 200 µl saline injected intraperitoneally twice, 2 wks apart. Two weeks after the second injection, 25 µl bupivacaine (0.375%, APP Pharmaceuticals) or 0.9% saline was injected at the sciatic and femoral nerves of the hind leg. Twenty minutes after the initial injection, animals were injected with labeled antigen in the dorsum of the hind paw.
Electrical Nerve Stimulation
Mice were anesthetized with isoflurane in the prone position. A 28-gauge uncoated grounding electrode (Technomed) was placed subcutaneously at the top of the thigh, and a coated needle electrode (Alpine Biomed) was inserted by the popliteal lymph node. Stimulation was delivered by a Biopac stimulation module controlled with AcqKnowledge 4.1 software. Parameters were −5V constant, 0.75 msec pulse duration, 20 Hz (50 msec period). Stimulation was applied for 5 min; after the first minute, KLH-800CW was injected into the dorsum of the hind paw.
Magnetic nerve Stimulation
Mice were anesthetized with isoflurane in the prone position. Magnetic stimulation was administered using an MC-B35 butterfly coil driven by a MagPro stimulator (Magventure) aimed at the popliteal and sciatic lymph nodes. The parameters used were 50% power, 120 pulses (2 Hz, 60 s) train 1, 1 s train interval. Control animals were anesthetized and the magnetic coil was positioned, but no current was applied. Immediately after stimulation, mice were removed from the table, then KLH-800CW was injected into the dorsum of the hind paw.
Antigen concentration compared with fluorescence and in situ compared with in-plate fluorescence were analyzed by linear regression. Control and experimental popliteal and sciatic lymph node signals were individually analyzed by unpaired Student t test. Antigen concentration curves and control/TRPV1/NaV1.8 lymph node fluorescence were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post-test. All tests with p < .05 were considered statistically significant. Statistical analyses were performed using Graphpad Prism 6 software. Unless otherwise stated, all numbers are given as average ± standard error of the mean; n represents the number of mice used in each group. In graphs, * indicates p < .05, ** indicates p < .01, *** indicates p < .001 and **** indicates p < .0001.
Antigen Flow Through the Lymphatic Chain Was Monitored with Infrared Fluorescent Dye-Labeled Antigen
Antigen Flow Through Peripheral Lymph Nodes Is Restricted in Immunized Animals
To assess the specificity of this restriction, mice were immunized with either KLH or OVA and then challenged with labeled KLH (KLH-800CW) or OVA (OVA-680LT). Immunization with KLH restricted the subsequent challenge with labeled KLH (Figure 2C, second panel, KLH in green) but failed to restrict an unrelated antigen, labeled OVA (Figure 2C, fourth panel, OVA in red). However, immunization with OVA led to subsequent restriction of OVA (Figure 2C, third panel, OVA in red). Together, these results indicate that an antigen-specific “memory” leads to subsequent restriction of the same antigen.
To determine the durability of the “memory,” mice were challenged with labeled KLH at various time points up to 17 wks following the second immunization. Control animals received immunization with saline. A consistent decrease in antigen concentration was observed in the sciatic lymph node in immunized mice as compared with the naïve (saline-immunized) controls. This indicates that immunization with an antigen confers a long-lasting restriction of that antigen upon subsequent challenge (Figure 2D).
Antigen Flow Restriction Is Dependent on Sensory neural Input
Direct binding of bacterial products to nociceptors induced hyperalgesia and modulated the local immune response, and ablation of NaV1.8-lineage neurons increased local immune infiltration and lymphadenopathy of the draining lymph node (9). To determine the role of nociceptors in modulating the transit of labeled antigen, we generated NaV1.8-Cre/diphtheria toxin A (DTA) mice to specifically ablate these cells. NaV1.8-Cre±DTA± mice are devoid of all NaV1.8-expressing nociceptors (15). Restriction of antigen flow to the sciatic lymph node after immunization was abolished in NaV1.8-Cre±DTA± mice, indicating that NaV1.8-lineage nociceptor neurons play a key role in modulating antigen flow through lymphatics (Figures 3C, D) (control, 15.41 ± 3.526, n = 13 versus NaV1.8-Cre±DTA±, 35.56 ± 6.035, n = 16, p < .05 by t test). No difference was observed in naïve NaV1.8-Cre±DTA± mice compared with controls (data not shown). Additionally, serum IgG levels against KLH were similar in NaV1.8-Cre±DTA± and control mice, suggesting that these animals were not merely immunocompromised (Figures 3E-F). These data suggest that neuronal input is required for the observed restriction of flow of immunized antigens.
Next we used targeted diphtheria toxin-based cell ablation to investigate whether transient receptor potential channel vanilloid 1 (TRPV1)-expressing neurons play a role in modulating antigen flow. TRPV1 is expressed by a subset of NaV1.8-expressing nociceptive neurons and is essential for transmitting nociceptive thermal stimuli and establishing and maintaining inflammatory hyperalgesia (16). TRPV1-Cre±DTA± mice and control littermates developed restriction of antigen trafficking to the sciatic lymph node. However, immunized NaV1.8-Cre±DTA± mice exhibited increased antigen concentration in the sciatic lymph node (Figure 3G) (control, 6.783 ± 1.290, n = 9; TRPV1-DTA, 6.345 ± 1.547, n = 10; NaV1.8-DTA, 43.93 ± 15.57, n = 6, p < .001 by one-way ANOVA Bonferroni post-test). These data indicate that a subset of NaV1.8 neurons that lack TRPV1 expression plays an important role in modulating antigen restriction.
Neuronal Stimulation Restricts Antigen Flow
We next used a time-varying magnetic field with an electromagnetic coil directed to the hind leg to stimulate the local neural network in naïve animals. Sham animals received no stimulation. Following stimulation, animals were subjected to challenge with KLH-800CW in the hind paw. Similar to the electrical stimulation, activation of the neural network using magnetic stimulation significantly restricted antigen flow to the sciatic lymph node in naïve animals as compared with sham stimulated animals (sham stimulated, 55.99 ± 4.993, n = 10 versus magnetically stimulated, 30.87 ± 4.169, n = 12, p < .001 by t test) (Figures 4C, D). These data clearly demonstrate that direct neuronal activation regulates antigen flow in the lymph nodes.
Neuronal Fc Receptors Play a Role in Antigen Restriction
To explore the consequences of deleting FcR expression, we analyzed antigen restriction in naïve and immunized FcR KO mice. Comparable levels of KLH signals were observed in naïve Balb/c and FcR KO mice, indicating that antigen trafficking is not different in naïve mice lacking FcR (Balb/c, 62.50 ± 5.614, n = 8 versus FCR KO, 71.48 ± 8.197, n = 6) (Figure 5B). In contrast, a significantly increased level of antigen was observed in the sciatic lymph nodes of KLH-immunized FcR KO compared with wild-type mice (Balb/c, 15.76 ± 2.721, n = 10 versus FCR KO, 44.29 ± 6.500, n = 11, p < .001 by t test) (Figure 5C). Moreover, in mice immunized with KLH, antigen and Fc receptors colocalized on PGP9.5 + ve neuronal tissue at the site of injection. (Figure 5D). Together, these data suggest that neuronal Fc receptors participate in the neural restriction of immunized antigen.
We and others have identified reflex neural circuits that modulate the output of the immune system in other organ systems. In Caenorhabditis elegans, neurons can regulate innate immunity, indicating the early evolutionary origins of the regulation of innate immune responses by the nervous system (19,20). In mammals, neural networks regulate immune responses in the spleen, gut, lungs, central nervous system and even cancer tissues (21, 22, 23, 24, 25). It has been proposed that neural activation in the lymphoid organs maintains the integrity of the antibody response (21), but a direct modulation of antigen transit, as revealed here, has not been described. Identification of a neural reflex mechanism that contributes to antigen transit reveals a previously unknown mechanism for antigen restriction. The observation that a depletion of NaV1.8+ neurons results in an abrogation of antigen restriction indicates that there is a role for nociceptor neurons in antigen recognition and restriction. Nociceptors have previously been linked to mediating bacteria-induced pain, modulating allergic inflammatory responses and potentiating adaptive immune responses (9,10,12). Here we show that nociceptors are crucial for antigen-specific restriction of antigen transport through the lymphatic system. Furthermore, our data provide direct evidence that expression of FcR is essential for mediating antigen-specific restriction through lymphatics. It has been argued that a subpopulation of nociceptive neurons expresses functional FcR, and that activation of this receptor by antigen-antibody complexes increases neuronal excitability (26). The current study suggests that antigen-specific activation of nociceptors via FcR contributes to antigen restriction and transport.
Our data reveal a potential mechanism to control the spread of pathogenic bacteria or micrometastases. Upon infection, bacteria routinely disseminate through the lymphatic system, eventually leading to a systemic infection. It is plausible to selectively activate the local neural network of the neurons innervating the draining lymph node to prevent bacteremia and sepsis. Upon activation, neurons convey information through temporal patterns of action potentials and graded membrane potential shifts (27, 28, 29). It is conceivable to record and decode the sensory signals activated during the antigen restriction reflex, which could have implications for developing novel therapies for bacterial infection. Indeed, distinct signals were recorded in the sciatic nerve of the immunized animals after antigen challenge in the foot compared with naïve animals (data not shown). The lymphatic system also plays a crucial role in cancer metastasis (30, 31, 32). Many therapeutic courses involve lymphatic mapping to determine which lymph nodes are draining the site of a primary tumor (33, 34, 35, 36, 37, 38). These lymph nodes are then monitored for development of new micrometastases. An improved understanding of the role of the neural network in modulating lymph node trafficking suggests that limiting micrometastases by activating the local neural network could provide a novel therapy for cancer metastasis.
The lymphatic system plays a vital role in conveying free antigen and antigen-presenting cells from the tissues to local draining lymph nodes for immune surveillance and activation. Lymphatic flow through the lymph vessels is modulated by the intrinsic contractile property of the lymphatic smooth muscles. Changes in the local microenvironment regulate the function of the smooth muscles and modulate the lymphatic flow. During critical illness, local and systemic inflammation play an important role in initiating and establishing muscle weakness (39, 40, 41). Although inflammatory responses induced by an antigen challenge in immunized animals can lead to a reduction in muscle contractions, resulting in restriction of lymphatic flow and antigen trafficking, these effects require chronic exposure to inflammatory mediators (39). Here, we monitored antigen transport in an acute setting. It is plausible that delayed type hypersensitivity (DTH) could lead to increased pain and immobility in an acute setting. However, these responses usually peak within hours. In animals with KLH-induced DTH responses, the peak swelling in response to KLH administration in the foot is observed after 24 h (42). In contrast, our data demonstrate that prior immunization with an antigen restricts antigen flow within minutes in an immunized animal, indicating that DTH does not mediate these responses. Moreover, direct electrical stimulation induced antigen restriction in naïve animals, further confirming the role of neural activation in modulating lymphatic function.
The discovery and characterization of a novel neural circuit wherein sensory and motor nerves regulate antigen trafficking from distal to proximal lymph nodes provides additional insight into the interplay between the nervous and immune systems. Ongoing studies to understand the sensory signals and molecular mechanisms of this antigen restriction reflex might reveal direct sensory and effector roles of the nervous system in antigen restriction and provide insights that have implications for developing bioelectronic therapies.
The authors declare that they have no competing interests as defined by Bioelectronic Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
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