Colin Reardon

Early-life enteric infection disrupts the developing microbiota–gut–brain (MGB) axis. Using a neonatal Enteropathogenic Escherichia coli (EPEC) model, we show that intestinal epithelial cell (IEC) nucleotide oligomerization domain (NOD)1 signaling coordinates neuroimmune outcomes throughout early development and into adulthood. Neonatal mice infected at postnatal day (P)7 exhibited ileal inflammation, with increased pro-inflammatory cytokines and monocyte/macrophage infiltration, and loss of myenteric neurons in wild-type mice but not in NOD1 conditional knockout (Nod1 ^ΔIEC ) mice. These deficits persisted into adulthood (P56), with increased intestinal permeability, sustained inflammatory/repair signatures, neuroinflammation, altered neurogenesis, and impaired recognition memory, which were absent in Nod1 ^ΔIEC mice, establishing a crucial role for IEC NOD1 in long-term MGB remodeling. Last, bioactive peptidoglycan fragments (muropeptides) were isolated from probiotic Lactobacillus strains and tested as possible therapeutics. Excitingly, supplementation of muropeptides attenuated neonatal EPEC-induced mucosal inflammation and enteric neuron loss without altering bacterial burden, potentially via IEC NOD2. Together, these findings identify IEC NOD-dependent signaling linking early-life enteric infection to enduring gut-brain dysfunction and reveal probiotic-derived muropeptides as candidate therapeutics.

Background: Infection with enteric bacterial pathogens continues to cause significant morbidity and mortality throughout the world. Effective control of these infections requires coordinated immune responses that are shaped in part by bidirectional communication between the nervous and immune systems. Sensory neurons are now well appreciated to detect inflammatory or microbial stimuli in numerous organ systems, including the gastrointestinal (GI) tract, and for eliciting the recruitment or activation of specific immune cells. Central to the host protective effect of these neuroimmune circuits is the localized release of neurotransmitters that activate substance P (SP) receptor signaling. However, the precise contribution of sensory neuropeptides encoded by the Tac1 gene, such as SP, in the host response to enteric infection remains incompletely understood. Methods: The role of the Tac1-encoded sensory neurotransmitters in the host response to Citrobacter rodentium was assessed using Tac1 ^-/- and wildtype (WT) mice. Infection and inflammation were determined by bacterial enumeration, histopathology, qPCR, and flow cytometry. Results: Tac1 ^-/- mice had significantly reduced pathogen shedding and colonic bacterial burden, accompanied by decreased expression of inflammatory cytokines and chemokines compared to WT controls. In accordance with reduced chemokine production, we observed reduced colonic recruitment of neutrophils, monocytes, and IFNγ- and IL-17A-producing T-cells in Tac1 ^-/- compared with WT mice. This reduced immune response was associated with markedly reduced colonic histopathology. Conclusions: Sensory neuropeptides encoded by Tac1 regulate key aspects of the immune response to enteric bacterial infection and may serve as unique targets in the treatment of enteric disease.

Bidirectional communication between the nervous and immune systems has been demonstrated to limit or enhance immune cell function across organ systems and conditions. Although these neuroimmune circuits can become activated as an anti-inflammatory reflex, vagus nerve stimulation (VNS) reduces inflammation in models of endotoxemia, rheumatoid arthritis, and intestinal inflammation. In the spleen during endotoxemia, VNS activates a “cholinergic anti-inflammatory pathway” (CAIP), whereby choline acetyltransferase (ChAT)-expressing T cells release acetylcholine to reduce macrophage activation. VNS can also drive CAIP-independent pathways to reduce inflammation in the spleen and intestinal tract, although the circuitry modulating colonic inflammation remains underexplored. Here, we demonstrate that left cervical VNS reduces acute LPS-induced inflammation, evidenced by reduced Tnfa expression in colon and spleen and decreased circulating TNFα. In the colon, these protective effects required efferent but not afferent VNS and were independent of ChAT+ T cells, IL10, β-adrenergic signaling, and colonic sympathetic innervation. Critically, the ability of VNS to modulate colonic inflammation depended on prior tissue-specific inflammation. Mice recovering from DSS colitis, despite near-complete histological recovery, were refractory to the protective effects of VNS in the colon. This lack of efficacy in the colon was not reflected in measures of inflammation in the spleen or serum, highlighting the need for target-organ-specific monitoring. This loss of efficacy after colonic inflammation was transient, with restoration occurring upon complete recovery. These findings demonstrate that VNS efficacy in colonic inflammation depends on circuitry distinct from canonical systemic anti-inflammatory pathways, and that tissue responsiveness is shaped by anatomical site and inflammatory history. Key Points: Electrical stimulation of the left cervical vagus nerve reduces LPS-induced inflammation in the mouse colon. This colonic anti-inflammatory effect requires vagal efferents but not afferent signaling. Unlike canonical splenic anti-inflammatory pathways, the colonic response does not require ChAT+ T cells, IL-10, β-adrenergic signaling, or local sympathetic innervation. Recent DSS colitis abolishes colonic responsiveness to VNS despite preserved splenic and systemic anti-inflammatory effects. Recovery of VNS anti-inflammatory efficacy after colitis shows that neuroimmune responsiveness in the colon is dynamically shaped by inflammatory history.

Vagus nerve stimulation (VNS) has been shown to limit immune cell activity across several pathologies ranging from sepsis to auto-immune diseases. While stimulation of vagal efferent neurons is known to reduce maladaptive host responses during endotoxemia, only selective vagal afferent neuron stimulation inhibited TLR7-induced macrophage activation and neutrophil recruitment to the lung. These anti-inflammatory actions are dependent on adrenal gland–derived epinephrine, as adrenalectomy or inhibition of epinephrine production eliminated the protection afforded by VNS. Selective afferent VNS induced activation in the nucleus tractus solitarius and the rostral ventrolateral medulla. Inhibition of neuronal activity in this brain region that controls peripheral sympathetic nervous system activity rendered VNS ineffective. Activation of the β ₂ -adrenergic receptor (β ₂ AR) is critical for innate immune cell suppression, as the anti-inflammatory effects of VNS were eliminated in β ₂ AR knockout mice and with pharmacological inhibition of the β ₂ AR. These findings demonstrate a previously unidentified neuroimmune circuit elicited by VNS that can control acute lung inflammation.

Background: Infection with enteric bacterial pathogens continues to cause significant morbidity and mortality throughout the world. These pathogens include enterohemorrhagic and enteropathogenic Escherichia coli , which transit the intestinal tract, efface microvilli, and attach firmly to intestinal epithelial cells predominantly in the colon. Investigation of these human-adapted pathogens has been greatly aided by mouse models of infection. The mouse-adapted attaching and effacing pathogen Citrobacter rodentium utilizes many similar mechanisms of pathogenesis, including the use of a type III secretion system, and virulence factors encoded in a locus of enterocyte effacement. Although this model has allowed for assessing the complexity of the host response, the complex interplay between the nervous and immune systems in response to infection remains incomplete. Methods: We assessed the role of sensory neurotransmitters encoded by the Tac1 gene in the host response to C. rodentium . Results: Tac1 -deficient mice had significantly reduced pathogen shedding and colonic bacterial burden, accompanied by decreased expression of inflammatory cytokines and chemokines. In accordance with reduced chemokine production, we observed reduced colonic recruitment of specific immune cell populations in Tac1-/- compared to WT mice. Conclusions: Sensory neuropeptides regulate key aspects of enteric bacterial infection and may serve as unique targets in the treatment of enteric disease.

Mucosal immune responses to enteric bacterial infections are highly coordinated processes that orchestrate host protection while minimizing the potential for immune-triggered pathology. In the intestinal tract, bidirectional communication occurs between the nervous and immune systems to affect local immune responses by modulating the activity of resident and recruited immune cells, and indirectly on the supporting stromal cells. These neuroimmune signaling pathways that alter host defense have focused on specialized sensory innervation and the unique neurotransmitters released from them. Although the sympathetic nervous system has been established to induce a tissue-protective phenotype in subpopulations of neuron-associated macrophages in the small intestine, the role of these neurons during enteric bacterial infection was unknown. Using genetic labeling of activated neurons with Arc TRAP, we demonstrate that colonic infection induces activation of the rostral ventrolateral medulla, a major sympathetic center in the brainstem. The importance of peripheral sympathetic neurons was further demonstrated using chemical sympathectomy that significantly increased bacterial burden during Citrobacter rodentium ( C. rodentium ) infection. Increased bacterial burden was matched by a deficit in host protection due to reduced IFNγ production by colonic CD4+ T-cells. Sympathectomy, however, did not diminish the capacity to differentiate into IFNγ- or IL-17A- producing T-cells in vitro , suggesting that the lack of sympathetic innervation during infection may alter this process in vivo without causing sustained T-cell intrinsic defects. In assessing which receptors could mediate these effects, pharmacological antagonists selective for α- adrenergic receptors (αAR), but not β-adrenergic receptors, increased bacterial burden and reduced colonic IFNγ production. Using isolated cell types from the colon of uninfected and infected mice, we identified the αΑR subtypes expressed on immune and stromal cells, with significant upregulation of these receptors on T-cells during C. rodentium infection. Together these data demonstrate the unique role of the sympathetic nervous system and αAR in mucosal immune responses against enteric bacterial pathogens.

Vagus nerve stimulation (VNS) has been shown to limit immune cell activity across several pathologies ranging from sepsis to auto-immune diseases. While stimulation of vagal efferent neurons has been previously demonstrated to reduce maladaptive host responses during endotoxemia, only selective stimulation of vagal afferent neurons was able to inhibit TLR7-induced macrophage activation and neutrophil recruitment in the lung. These anti-inflammatory actions are facilitated by systemic increases in epinephrine, as VNS significantly increased epinephrine in the serum and bronchoalveolar lavage fluid, and inhibition of epinephrine production eliminated the protection afforded by VNS. Selective afferent VNS induced activation in the nucleus tractus solitarius and the rostral ventrolateral medulla. Inhibition of neuronal activity in this brain region that controls peripheral sympathetic nervous system activity rendered VNS ineffective. Activation of the β ₂ -adrenergic receptor (β ₂ AR) is critical for innate immune cell suppression, as the anti-inflammatory effects of VNS were eliminated in β ₂ AR-knock out mice, and with pharmacological inhibition of the β ₂ AR. Analysis of the immune cells responding to R848 critically identified that plasmacytoid dendritic cells were refractive to inhibition by VNS, and this corresponded to lack of β ₂ AR expression. These findings demonstrate a novel neuro-immune circuit elicited by VNS that can control acute lung inflammation. Summary: We have identified a novel neuro-immune circuit activated by afferent vagus nerve stimulation to reduce acute lung inflammation. This effect was dependent on vagal-induced adrenal gland-derived epinephrine release that initiates anti-inflammatory β2-adrenergic receptor signaling in innate immune cells within the lung.

Immune responses in the intestine are intricately balanced to prevent pathogen entry without inducing immunopathology. The nervous system is well established to interface with the immune system to fine-tune immunity in various organ systems including the gastrointestinal tract. Specialized sensory neurons can detect bacteria, bacterial products, and the resulting inflammation, to coordinate the immune response in the gastrointestinal tract. These sensory neurons release peptide neurotransmitters such as Substance P (SP), to induce both neuronal signaling and localized responses in nonneuronal cells. With this in mind, we assessed the immunoregulatory roles of SP receptor signaling during enteric bacterial infection with the noninvasive pathogen Citrobacter rodentium . Pharmacological antagonism of the SP receptor significantly reduced bacterial burden and prevented colonic crypt hyperplasia. Mice with SP receptor signaling blockade had significantly reduced inflammation and recruitment of T cells in the colon. Reduced colonic T cell recruitment is due to reduced expression of adhesion molecules on colonic endothelial cells in SP receptor antagonist-treated mice. Using SP receptor T cell conditional knockout mice, we further confirmed SP receptor signaling enhanced select aspects of T cell responses. Our data demonstrate that SP receptor signaling can significantly reduce inflammation and prevent host-maladaptive responses without impinging upon host protection.