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.
Abstract Mammals store memories in the nervous and immune systems. Sensory neurons have been implicated in enhancing neurological memory, but whether neurons participate during immunity to novel antigens is unknown. Here, mice rendered deficient in transient receptor potential vanilloid 1 (TRPV1)-expressing sensory neurons, termed “nociceptors,” fail to develop competent antibody responses to KLH and hapten-NP. Moreover, selective optogenetic stimulation of TRPV1 neurons during immunization significantly enhanced antibody responses to antigens. Thus, TRPV1 nociceptors mediate antibody responses to novel antigen, and stimulating TRPV1 nociceptors enhances antibody responses during immunization. This is the first genetic and selective functional evidence that nociceptors are required during immunization to produce antigen-specific antibodies. Summary The first genetic and selective functional evidence showing that TRPV1-expressing nociceptors are required for competent antibody responses to novel antigen, and stimulating TRPV1 nociceptors enhances antibody responses to novel antigen.
Inflammatory conditions characterized by excessive peripheral immune responses are associated with diverse alterations in brain function, and brain-derived neural pathways regulate peripheral inflammation. Important aspects of this bidirectional peripheral immune-brain communication, including the impact of peripheral inflammation on brain region-specific cytokine responses, and brain cholinergic signaling (which plays a role in controlling peripheral cytokine levels), remain unclear. To provide insight, we studied gene expression of cytokines, immune cell markers and brain cholinergic system components in the cortex, cerebellum, brainstem, hippocampus, hypothalamus, striatum and thalamus in mice after an intraperitoneal lipopolysaccharide injection. Endotoxemia was accompanied by elevated serum levels of interleukin (IL)-1β, IL-6 and other cytokines and brain region-specific increases in Il1b (the highest increase, relative to basal level, was in cortex; the lowest increase was in cerebellum) and Il6 (highest increase in cerebellum; lowest increase in striatum) mRNA expression. Gene expression of brain Gfap (astrocyte marker) was also differentially increased. However, Iba1 (microglia marker) mRNA expression was decreased in the cortex, hippocampus and other brain regions in parallel with morphological changes, indicating microglia activation. Brain choline acetyltransferase (Chat) mRNA expression was decreased in the striatum, acetylcholinesterase (Ache) mRNA expression was decreased in the cortex and increased in the hippocampus, and M1 muscarinic acetylcholine receptor (Chrm1) mRNA expression was decreased in the cortex and the brainstem. These results reveal a previously unrecognized regional specificity in brain immunoregulatory and cholinergic system gene expression in the context of peripheral inflammation and are of interest for designing future antiinflammatory approaches.
Abstract Chronic sepsis survival is still an unchartered territory, where system-wide, sometimes perennial dysfunction occurs, usually with dire consequences. In the present study, polimicrobial abdominal sepsis -or sham surgery- was induced using the CLP model. Long-term survivors (>7 days) were sacrificed at weekly intervals, and blood and spleen were collected. Splenic and circulating leukocyte populations were analyzed by flow cytometry. Cytokines were measured by bead-array assay. We found an early increase in splenic size and weight, followed by massive, significant influx of granulocytes, monocytes, and B-cells. Splenic monocytes and granulocytes remained high until week 4. In contrast, splenic T-cell populations remained stable after sepsis, and B-cells returned to baseline by week 3. In peripheral circulation, we found early increases in IL-6 (25-fold; p=0.01), and TNF (7-fold; p=0.01). Other cytokines, including IL-2, IFN-γ, and IL-17A were not different between groups. Serum IL-6 concentration dropped back to levels similar to those of sham mice by week 4. Strikingly, around this time all surviving mice developed spontaneous abdominal abscesses. This is interesting since IL-6 levels have been shown to be a good predictor of early mortality. The fact that a late-phase decrease may be related to a the development of a state of imunosuppression opens new avenues to understanding CARS syndrome/immunoparalysis state known to be devastating in long term septic patients.
Abstract Severe sepsis accounts for over 200,000 deaths in US annually (Angus et al, CCM, 2001). The mechanism involves endogenous molecules that impair organ function. Extracellular hemoglobin is one such factor enhancing tissue damage during sepsis. We designed haptoglobin-coated beads for a perfusion system to remove hemoglobin in an experimental sepsis model. Surprisingly, we observed that in addition to removing hemoglobin, haptoglobin beads also captured large amounts of HMGB1. HMGB1 is a major mediator on the final common pathway to lethality in sepsis (Yang et al, BBA 2009). Addition of haptoglobin suppressed HMGB1-stimulated TNF and IL-8 release in cultured macrophages. Haptoglobin knockout mice subjected to cecal perforation-induced sepsis had significantly higher serum HMGB1 levels and higher mortality compared to wild type animals (75% survival in wild type vs. 34% in haptoglobin knockout mice; n=15/group, P<0.05). Treatment with anti-HMGB1 antibodies significantly reduced the mortality in haptoglobin knockout mice (12% survival in control vs. 56% in animals receiving HMGB1 antibodies; n=16/group, P<0.05). Structure-function analysis revealed that haptoglobin beta subunit alone is sufficient to recapitulate the protective effects observed with haptoglobin. These findings indicate that haptoglobin is an endogenous modulator of HMGB1 that is capable of reducing the toxicity of HMGB1 in sepsis. Supported in part by grants from NIH (RO1GM62508, to KJT and RO1GM098446, to HY).
Abstract Severe sepsis, a syndrome complicating infection or injury, is a leading cause of death. It also has long-term deleterious effects on survivors, including high mortality and diminished quality of life. High-mobility group box 1 (HMGB1) is a critical late mediator of acute sepsis that remains elevated after other inflammatory cytokines have normalized. Here we studied the kinetics of circulating HMGB1 in mice surviving severe abdominal sepsis, and the effects of administering neutralizing anti HMGB1 mAb. In a standardized model of murine sepsis, we observed that mice surviving sepsis developed splenomegaly for at least four weeks. Splenocytes from sepsis survivors produced significantly more cytokines following exposure to TLR ligands. Circulating HMGB1 levels are significantly increased for at least 4 weeks after sepsis. Treatment with anti-HMGB1 neutralizing monoclonal antibodies effectively prevented the development of splenomegaly, leukocytosis and anemia. Chronic administration of recombinant HMGB1 to healthy mice induced significant splenomegaly and leukocytosis. Recombinant HMGB1 also sensitized splenocytes and significantly increased the release of TNF and IL-6. In conclusion, serum HMGB1 levels are persistently elevated for at least 4 weeks in mice that survive lethal sepsis. Anti-HMGB1 mAb administration reverses the development of splenomegaly, leukocytosis and anemia, which may have implications for understanding the pathogenesis of post-sepsis complications.
The vagus nerve plays an important role in the regulation of organ function, including reflex pathways that regulate immunity and inflammation. Recent studies using genetically modified mice have improved our understanding of molecular mechanisms in the neural control of immunity. However, mapping neural signals transmitted in the vagus nerve in mice has been limited by technical challenges. Here, we have standardized an experimental protocol to record compound action potentials transmitted in the vagus nerve.The vagus nerve was isolated in Balb/c and B6.129S mice, and placed either on a hook or cuff electrode. The electrical signals from the vagus nerve were digitized using either a Neuralynx or Plexon data acquisition system. Changes in the vagus nerve activity in response to anesthesia, feeding and administration of bacterial endotoxin were analyzed.We have developed an electrophysiological recording system to record compound action potentials from the cervical vagus nerve in mice. Cuff electrodes significantly reduce background noise and increase the signal to noise ratio as compared to hook electrodes. Baseline vagus nerve activity varies in response to anesthesia depth and food intake. Analysis of vagus neurograms in different mouse strains (Balb/c and C57BL/6) reveal no significant differences in baseline activity. Importantly, vagus neurogramactivity in wild type and TLR4 receptor knock out mice exhibits receptor dependency of endotoxin mediated signals.These methods for recording vagus neurogram in mice provide a useful tool to further delineate the role of vagus neural pathways in a standardized murine disease model.
Introduction Inflammation is an inherently self-amplifying process, resulting in progressive tissue damage when unresolved. A brake on this positive feedback system is provided by the nervous system which has evolved to detect inflammatory signals and respond by activating anti-inflammatory processes, including the cholinergic anti-inflammatory pathway mediated by the vagus nerve. Acute pancreatitis, a common and serious condition without effective therapy, develops when acinar cell injury activates intrapancreatic inflammation. Prior study has shown that electrical stimulation of the carotid sheath, which contains the vagus nerve, boosts the endogenous anti-inflammatory response and ameliorates acute pancreatitis, but it remains unknown whether these anti-inflammatory signals originate in the brain. Methods Here, we used optogenetics to selectively activate efferent vagus nerve fibers originating in the brainstem dorsal motor nucleus of the vagus (DMN) and evaluated the effects on caerulein-induced pancreatitis. Results Stimulation of the cholinergic neurons in the DMN significantly attenuates the severity of pancreatitis as indicated by reduced serum amylase, pancreatic cytokines, tissue damage, and edema. Either vagotomy or silencing cholinergic nicotinic receptor signaling by pre-administration of the antagonist mecamylamine abolishes the beneficial effects. Discussion These results provide the first evidence that efferent vagus cholinergic neurons residing in the brainstem DMN can inhibit pancreatic inflammation and implicate the cholinergic anti-inflammatory pathway as a potential therapeutic target for acute pancreatitis.
