Yaakov A. Levine

Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit

A neural circuit that involves a specialized population of memory T cells regulates the immune response. Neural circuits regulate cytokine production to prevent potentially damaging inflammation. A prototypical vagus nerve circuit, the inflammatory reflex, inhibits tumor necrosis factor–α production in spleen by a mechanism requiring acetylcholine signaling through the α7 nicotinic acetylcholine receptor expressed on cytokine-producing macrophages. Nerve fibers in spleen lack the enzymatic machinery necessary for acetylcholine production; therefore, how does this neural circuit terminate in cholinergic signaling? We identified an acetylcholine-producing, memory phenotype T cell population in mice that is integral to the inflammatory reflex. These acetylcholine-producing T cells are required for inhibition of cytokine production by vagus nerve stimulation. Thus, action potentials originating in the vagus nerve regulate T cells, which in turn produce the neurotransmitter, acetylcholine, required to control innate immune responses.

Rethinking inflammation: neural circuits in the regulation of immunity

Summary:  Neural reflex circuits regulate cytokine release to prevent potentially damaging inflammation and maintain homeostasis. In the inflammatory reflex, sensory input elicited by infection or injury travels through the afferent vagus nerve to integrative regions in the brainstem, and efferent nerves carry outbound signals that terminate in the spleen and other tissues. Neurotransmitters from peripheral autonomic nerves subsequently promote acetylcholine‐release from a subset of CD4+ T cells that relay the neural signal to other immune cells, e.g. through activation of α7 nicotinic acetylcholine receptors on macrophages. Here, we review recent progress in the understanding of the inflammatory reflex and discuss potential therapeutic implications of current findings in this evolving field.

Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis

Significance Rheumatoid arthritis (RA) is a chronic, prevalent, and disabling autoimmune disease that occurs when inflammation damages joints. Recent advances in neuroscience and immunology have mapped neural circuits that regulate the onset and resolution of inflammation. In one circuit, termed “the inflammatory reflex,” action potentials transmitted in the vagus nerve inhibit the production of tumor necrosis factor (TNF), an inflammatory molecule that is a major therapeutic target in RA. Although studied in animal models of arthritis and other inflammatory diseases, whether electrical stimulation of the vagus nerve can inhibit TNF production in humans has remained unknown. The positive mechanistic results reported here extend the preclinical data to the clinic and reveal that vagus nerve stimulation inhibits TNF and attenuates disease severity in RA patients. Rheumatoid arthritis (RA) is a heterogeneous, prevalent, chronic autoimmune disease characterized by painful swollen joints and significant disabilities. Symptomatic relief can be achieved in up to 50% of patients using biological agents that inhibit tumor necrosis factor (TNF) or other mechanisms of action, but there are no universally effective therapies. Recent advances in basic and preclinical science reveal that reflex neural circuits inhibit the production of cytokines and inflammation in animal models. One well-characterized cytokine-inhibiting mechanism, termed the “inflammatory reflex,” is dependent upon vagus nerve signals that inhibit cytokine production and attenuate experimental arthritis severity in mice and rats. It previously was unknown whether directly stimulating the inflammatory reflex in humans inhibits TNF production. Here we show that an implantable vagus nerve-stimulating device in epilepsy patients inhibits peripheral blood production of TNF, IL-1β, and IL-6. Vagus nerve stimulation (up to four times daily) in RA patients significantly inhibited TNF production for up to 84 d. Moreover, RA disease severity, as measured by standardized clinical composite scores, improved significantly. Together, these results establish that vagus nerve stimulation targeting the inflammatory reflex modulates TNF production and reduces inflammation in humans. These findings suggest that it is possible to use mechanism-based neuromodulating devices in the experimental therapy of RA and possibly other autoimmune and autoinflammatory diseases.

α7 Nicotinic Acetylcholine Receptor Signaling Inhibits Inflammasome Activation by Preventing Mitochondrial DNA Release

The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Cellular stress is associated with changes in immunity and activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, a key component of innate immunity. Here we show that α7 nicotinic acetylcholine receptor (α7 nAchR)-signaling inhibits inflammasome activation and prevents release of mitochondrial DNA, an NLRP3 ligand. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibits inflammasome activation, whereas genetic deletion of α7 nAchR significantly enhances inflammasome activation. Acetylcholine accumulates in macrophage cytoplasm after adenosine triphosphate (ATP) stimulation in an α7 nAchR-independent manner. Acetylcholine significantly attenuated calcium or hydrogen oxide-induced mitochondrial damage and mitochondrial DNA release. Together, these findings reveal a novel neurotransmitter-mediated signaling pathway: acetylcholine translocates into the cytoplasm of immune cells during inflammation and inhibits NLRP3 inflammasome activation by preventing mitochondrial DNA release.

α7 nicotinic acetylcholine receptor (α7nAChR) expression in bone marrow-derived non-T cells is required for the inflammatory reflex.

The immune response to infection or injury coordinates host defense and tissue repair, but also has the capacity to damage host tissues. Recent advances in understanding protective mechanisms have found neural circuits that suppress release of damaging cytokines. Stimulation of the vagus nerve protects from excessive cytokine production and ameliorates experimental inflammatory disease. This mechanism, the inflammatory reflex, requires the α7 nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel expressed on macrophages, lymphocytes, neurons and other cells. To investigate cell-specific function of α7nAChR in the inflammatory reflex, we created chimeric mice by cross-transferring bone marrow between wild-type (WT) and α7nAChR-deficient mice. Deficiency of α7nAChR in bone marrow-derived cells significantly impaired vagus nerve-mediated regulation of tumor necrosis factor (TNF), whereas α7nAChR deficiency in neurons and other cells had no significant effect. In agreement with recent work, the inflammatory reflex was not functional in nude mice, because functional T cells are required for the integrity of the pathway. To investigate the role of T-cell α7nAChR, we adoptively transferred α7nAChR-deficient or WT T cells to nude mice. Transfer of WT and α7nAChR-deficient T cells restored function, indicating that α7nAChR expression on T cells is not necessary for this pathway. Together, these results indicate that α7nAChR expression in bone marrow-derived non-T cells is required for the integrity of the inflammatory reflex.

HMGB1 mediates splenomegaly and expansion of splenic CD11b+ Ly‐6Chigh inflammatory monocytes in murine sepsis survivors

More than 500,000 hospitalized patients survive severe sepsis annually in the USA. Recent epidemiological evidence, however, demonstrated that these survivors have significant morbidity and mortality, with 3‐year fatality rates higher than 70%. To investigate the mechanisms underlying persistent functional impairment in sepsis survivors, here we developed a model to study severe sepsis survivors following cecal ligation and puncture (CLP).

Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes.

Secreted by activated cells or passively released by damaged cells, extracellular HMGB1 is a prototypical damage-associated molecular pattern (DAMP) inflammatory mediator. During the course of developing extracorporeal approaches to treating injury and infection, we inadvertently discovered that haptoglobin, the acute phase protein that binds extracellular hemoglobin and targets cellular uptake through CD163, also binds HMGB1. Haptoglobin-HMGB1 complexes elicit the production of antiinflammatory enzymes (heme oxygenase-1) and cytokines (e.g., IL-10) in WT but not in CD163-deficient macrophages. Genetic disruption of haptoglobin or CD163 expression significantly enhances mortality rates in standardized models of intra-abdominal sepsis in mice. Administration of haptoglobin to WT and to haptoglobin gene-deficient animals confers significant protection. These findings reveal a mechanism for haptoglobin modulation of the inflammatory action of HMGB1, with significant implications for developing experimental strategies targeting HMGB1-dependent inflammatory diseases.

Spinal p38 MAP kinase regulates peripheral cholinergic outflow

The central nervous system (CNS) interacts with the periphery to provide feedback regulation during inflammation. The brain and spinal cord sense danger signals released by pathogens outside the CNS and activate wide-ranging defense mechanisms, such as fever and the hypothalamic–pituitary–adrenal axis. Under conditions of autoimmunity the CNS can also modulate somatic responses. For instance, p38 MAP kinase inhibition in the CNS reduces inflammation and bone destruction in rat adjuvant arthritis (1). The spinal mechanisms of this phenomenon have been well delineated (1,2) but the efferent neurologic connections remain poorly characterized. Direct stimulation of the vagus nerve suppresses systemic production of cytokines implicated in arthritis, such as tumor necrosis factor, interleukin-1, and interleukin-6, via a cholinergic antiinflammatory pathway (3). We therefore postulated that spinal p38 engages this pathway. To test this possibility, we used power spectral analysis of heart rate variability to quantify vagal output after intrathecal administration of a p38 inhibitor. The high-frequency power spectral component of heart rate variability (HFP) is a widely used parameter of parasympathetic activity that directly correlates with activation of the cholinergic antiinflammatory pathway (4,5). All animals were handled according to US Department of Agriculture guidelines, and procedures were reviewed and approved by the institutional animal subjects committee. Rats were anesthetized with isoflurane and placed in a supine dorsal recumbent position. Unipolar platinum electrodes were placed onto the anterior chest wall and attached to an amplifier (ECG 100C; Biopac Systems, Santa Barbara, CA). Data were recorded for 70 minutes at a sampling rate of 1 kHz. Power spectral analysis of heart rate variability was calculated using Acqknowledge software (Biopac Systems). After performing fast Fourier transform, the HFP was calculated using a frequency range adapted to the physiologic setting in rats (0.6–1 Hz), to quantify cholinergic outflow. We initially used a cholinesterase inhibitor, galanthamine, to verify that we could detect cholinergic changes in HFP. Vagal efferent fibers augment HFP by releasing acetylcholine, which binds to muscarinic receptors expressed by cardiac pacemaker cells. Acetylcholine is then rapidly converted into inactive metabolites by cholinesterases that can be inhibited with galanthamine. Galanthamine modulates this process by inhibiting acetylcholine esterase and through a central excitatory effect on vagal outflow (5). As shown in Figure 1A, rats treated with galanthamine exhibited significantly increased HFP. The effect of galanthamine on HFP was comparable with the level of vagal stimulation that produces antiinflammatory effects in septic shock models (5). This degree of pharmacologic or electrical vagal stimulation can reduce paw inflammation (6). Galanthamine can mimic the antiinflammatory effects of the vagus nerve, presumably through a combination of increased acetylcholine release and decreased esterase function (3,7). Figure 1 A, Systemic administration of the cholinesterase inhibitor galanthamine increases the cholinergic antiinflammatory pathway. The high-frequency component of heart rate variability (HFP), a measure of cholinergic vagal activity, was recorded for 5-minute ... We next evaluated the antiinflammatory effect of galanthamine. Figure 1B shows that the compound significantly reduced swelling in the rat carrageenan paw edema model (6). This observation supports the notion of a potential link between increases in HFP and peripheral antiinflammatory effects, although the relationship between the intensity of cholinergic stimulation in inflamed paws and that in normal cardiac tissue is not known. We then investigated whether p38 within the CNS controls the vagal cholinergic outflow, using the same HFP measurement protocol. Isoflurane-anesthetized Lewis rats (200–250 gm) were implanted with an intrathecal catheter as previously described (2). After a 6-day recovery period, HFP was measured before and after injection of 8 μg of SB203580 through the intrathecal catheter. Intrathecal SB203580 caused a 3-fold rise in HFP that was not observed with intrathecal saline (P < 0.001 by 2-way analysis of variance) (Figure 1C). The increase in cholinergic outflow was noted within 5 minutes and persisted for at least 1 hour after injection. The concentration of SB203580 used has been shown to reduce arthritis after intrathecal administration in the rat adjuvant arthritis model (1). It is a small fraction of the doses typically required to suppress arthritis when given systemically (8), suggesting that the effect is mediated by the CNS. Additional mechanistic studies are needed to determine whether the intensity of acetylcholine receptor stimulation in the synovium correlates with HFP. In conclusion, we found that a very small dose of a p38 inhibitor delivered locally to the intrathecal space increased HFP in the rat. This novel observation demonstrates that MAP kinase signaling within the CNS can potentially activate the cholinergic antiinflammatory pathway. Because vagal stimulation of similar intensity can block inflammation during septic shock (5), this pathway could contribute to the suppression of synovitis after spinal p38 blockade (1). While the peripheral cholinergic mechanisms are still being explored, some studies implicate the alpha7 cholinergic receptor. For instance, acetylcholine can reduce inflammation by stimulating alpha7 receptors on macrophages (7) and can decrease the production of cytokines and chemokines by synoviocytes in vitro (9). If this receptor is confirmed as a key link between the CNS and peripheral inflammation, then selective agonists could have therapeutic utility. Since HFP is a recognized marker of parasympathetic activity in humans (4), our findings using this model suggest that HFP could represent a useful measure in clinical studies of therapies aimed at modulating inflammation via the CNS.

Sequestering HMGB1 via DNA-Conjugated Beads Ameliorates Murine Colitis

Inflammatory bowel disease (IBD) is chronic inflammation of the gastrointestinal tract that affects millions of people worldwide. Although the etiology of IBD is not clear, it is known that products from stressed cells and enteric microbes promote intestinal inflammation. High mobility group box 1 (HMGB1), originally identified as a nuclear DNA binding protein, is a cytokine-like protein mediator implicated in infection, sterile injury, autoimmune disease, and IBD. Elevated levels of HMGB1 have been detected in inflamed human intestinal tissues and in feces of IBD patients and mouse models of colitis. Neutralizing HMGB1 activity by administration of anti-HMGB1 antibodies or HMGB1-specific antagonist improves clinical outcomes in animal models of colitis. Since HMGB1 binds to DNA with high affinity, here we developed a novel strategy to sequester HMGB1 using DNA immobilized on sepharose beads. Screening of DNA-bead constructs revealed that B2 beads, one linear form of DNA conjugated beads, bind HMGB1 with high affinity, capture HMGB1 ex vivo from endotoxin-stimulated RAW 264.7 cell supernatant and from feces of mice with colitis. Oral administration of B2 DNA beads significantly improved body weight, reduced colon injury, and suppressed colonic and circulating cytokine levels in mice with spontaneous colitis (IL-10 knockout) and with dextran sulfate sodium-induced colitis. Thus, DNA beads reduce inflammation by sequestering HMGB1 and may have therapeutic potential for the treatment of IBD.

Neurostimulation of the Cholinergic Antiinflammatory Pathway in Rheumatoid Arthritis and Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) and rheumatoid arthritis (RA) cause significant morbidity and mortality. Despite significant therapeutic advances, the medical need for patients with these disorders remains high. An important neural-immune regulatory mechanism termed the “inflammatory reflex,” and its efferent arm, the “cholinergic antiinflammatory pathway” regulate innate and adaptive immunity. An emerging body of evidence indicates that stimulation of this pathway with implantable medical devices is a feasible therapeutic approach in disorders of dysregulated inflammation. Herein we describe the underlying biology and the preclinical experiments done in standard animal models that provided the rationale for testing in clinical trials. The preclinical development approach comprised elements of classic drug and medical device development, yet had unique features and challenges. “Bioelectronic medicines” having ideal characteristics of both drugs and medical devices hold great conceptual promise for treatment of systemic diseases in the future. However studies being done today will help determine whether neurostimulation of the cholinergic antiinflammatory pathway (NCAP) has the potential in the nearer term to fulfill the needs of patients, caregivers and payers for an additional potential treatment option for inflammatory disorders, and might thus become one of the first feasible examples of a bioelectronic medicine.