Summary Proper adaptation to environmental perturbations is essential for tissue homeostasis. In the intestine, diverse environmental cues can be sensed by immune cells, which must balance resistance to microorganisms with tolerance, avoiding excess tissue damage. By applying imaging and transcriptional profiling tools, we interrogated how distinct microenvironments in the gut regulate resident macrophages. We discovered that macrophages exhibit a high degree of gene-expression specialization dependent on their proximity to the gut lumen. Lamina propria macrophages (LpMs) preferentially expressed a pro-inflammatory phenotype when compared to muscularis macrophages (MMs), which displayed a tissue-protective phenotype. Upon luminal bacterial infection, MMs further enhanced tissue-protective programs, and this was attributed to swift activation of extrinsic sympathetic neurons innervating the gut muscularis and norepinephrine signaling to β2 adrenergic receptors on MMs. Our results reveal unique intra-tissue macrophage specialization and identify neuro-immune communication between enteric neurons and macrophages that induces rapid tissue-protective responses to distal perturbations.

SUMMARY Intestinal peristalsis is a dynamic physiologic process influenced by dietary and microbial changes. It is tightly regulated by complex cellular interactions; however, our understanding of these controls is incomplete. A distinct population of macrophages is distributed in the intestinal muscularis externa. We demonstrate that in the steady state muscularis macrophages regulate peristaltic activity of the colon. They change the pattern of smooth muscle contractions by secreting bone morphogenetic protein 2 (BMP2), which activates BMP receptor (BMPR) expressed by enteric neurons. Enteric neurons, in turn, secrete colony stimulatory factor 1 (CSF1), a growth factor required for macrophage development. Finally, stimuli from microbial commensals regulate BMP2 expression by macrophages and CSF1 expression by enteric neurons. Our findings identify a plastic, microbiota-driven, crosstalk between muscularis macrophages and enteric neurons, which controls gastrointestinal motility.

The intestinal immune system has the challenging task of tolerating foreign nutrients and the commensal microbiome, while excluding or eliminating ingested pathogens. Failure in such balance leads to severe diseases such as inflammatory bowel diseases (IBD), food allergies or invasive gastrointestinal infections 1 . Multiple immune mechanisms are therefore in place to maintain tissue integrity, including balanced generation of effector T (T H ) cells and FOXP3 + regulatory T (pTreg) cells, which mediate resistance to pathogens and regulate excessive immune activation, respectively 1 – 4 . The gut–draining lymph nodes (gLNs) are critical sites for orchestrating adaptive immunity to luminal perturbations 5 – 7 . However, how they manage to simultaneously support tolerogenic and inflammatory reactions is incompletely understood. Here we report that gLNs are immunologically unique according to the functional gut segment they drain. Stromal and dendritic cell gene signatures as well as T cell polarization against the same luminal antigen differed between gLNs, the proximal small intestine–draining gLNs preferentially giving rise to tolerogenic and the distal gLNs to pro-inflammatory T cell responses. This segregation permitted targeting distal gLNs for vaccination and maintenance of duodenal pTreg cell induction during colonic infection. Conversely, the compartmentalized dichotomy was perturbed by surgical removal of select distal gLNs and duodenal infection, impacting both lymphoid organ and tissue immune responses. Our findings reveal that the conflict between tolerogenic and inflammatory intestinal responses is in part resolved by discrete gLN drainage, and encourage gut segment-specific antigen targeting for therapeutic immune modulation.

Transcranial direct current stimulation (tDCS) is a method for modulating cortical excitability by weak constant electrical current that is applied through scalp electrodes. Although often described in terms of anodal or cathodal stimulation, depending on which scalp electrode pole is proximal to the cortical region of interest, it is the orientation of neuronal structures relative to the direct current (DC) vector that determines the effect of tDCS. To investigate the contribution of neural pathway orientation, we studied DCS-mediated neuromodulation in an in vitro rat hippocampal slice preparation. We examined the contribution of dendritic orientation to the direct current stimulation (DCS) neuromodulatory effect by recording field excitatory postsynaptic potentials (fEPSPs) in apical and basal dendrites of CA1 neurons within a constant DC field. In addition, we assessed the contribution of axonal orientation by recording CA1 and CA3 apical fEPSPs generated by stimulation of oppositely oriented Schaffer collateral and mossy fiber axons, respectively, during DCS. Finally, nonsynaptic excitatory signal propagation was measured along antidromically stimulated CA1 axons at different DCS amplitudes and polarity. We find that modulation of both the fEPSP and population spike depends on axonal orientation relative to the electric field vector. Axonal orientation determines whether the DC field is excitatory or inhibitory and dendritic orientation affects the magnitude, but not the overall direction, of the DC effect. These data suggest that tDCS may oppositely affect neurons in a stimulated cortical volume if these neurons are excited by oppositely orientated axons in a constant electrical field.

Highlights d Enteric pathogens trigger reversible neuronal loss and longterm GI symptoms d Enteric infection-triggered neuronal loss is Nlrp6and caspase 11-dependent d Intestinal muscularis macrophages (MMs) rapidly respond to enteric pathogens d Neuronal death is limited by a MM-b 2 -adrenergic-arginase 1polyamine axis

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Objectives-To approximate methods for human transcranial magnetic stimulation (TMS) in rats, we tested whether lateralized cortical stimulation resulting in selective activation of one forelimb contralateral to the site of stimulation could be achieved by TMS in the rat.Methods-Motor evoked potentials (MEP) were recorded from the brachioradialis muscle bilaterally in adult male anesthetized rats (n=13). A figure-of-eight TMS coil was positioned lateral to midline. TMS intensity was increased stepwise from subthreshold intensities to maximal machine output in order to generate input-output curves and to determine the motor threshold (MT) for brachioradialis activation.Results-In 100% of the animals, selective activation of the contralateral brachiradialis, in the absence of ipsilateral brachiradialis activation was achieved, and the ipsilateral brachioradialis was activated only at TMS intensities exceeding contralateral forelimb MT. With increasing TMS intensity, the amplitudes of both the ipsilateral and contrlateral signals increased in proportion to TMS strength. However the input-output curves for the contralateral and ipsilateral brachioradialis were significantly different (p<0.001) such that amplitude of the ipsilateral MEP was reliably lower than the contralateral signal.Conclusions-We demonstrate that lateralized TMS leading to asymmetric brachioradialis activation is feasible with conventional TMS equipment in anesthetized rats.Significance-These data show that TMS can be used to assess the unilateral excitability of the forelimb descending motor pathway in the rat, and suggest that rat TMS protocols analogous to human TMS may be applied in future translational research.