PrestoCell: A persistence-based clustering approach for rapid and robust segmentation of cellular morphology in three-dimensional data.

Light microscopy methods have continued to advance allowing for unprecedented analysis of various cell types in tissues including the brain. Although the functional state of some cell types such as microglia can be determined by morphometric analysis, techniques to perform robust, quick, and accurate measurements have not kept pace with the amount of imaging data that can now be generated. Most of these image segmentation tools are further burdened by an inability to assess structures in three-dimensions. Despite the rise of machine learning techniques, the nature of some biological structures prevents the training of several current day implementations. Here we present PrestoCell, a novel use of persistence-based clustering to segment cells in light microscopy images, as a customized Python-based tool that leverages the free multidimensional image viewer Napari. In evaluating and comparing PrestoCell to several existing tools, including 3DMorph, Omipose, and Imaris, we demonstrate that PrestoCell produces image segmentations that rival these solutions. In particular, our use of cell nuclei information resulted in the ability to correctly segment individual cells that were interacting with one another to increase accuracy. These benefits are in addition to the simplified graphically based user refinement of cell masks that does not require expensive commercial software licenses. We further demonstrate that PrestoCell can complete image segmentation in large samples from light sheet microscopy, allowing quantitative analysis of these large datasets. As an open-source program that leverages freely available visualization software, with minimum computer requirements, we believe that PrestoCell can significantly increase the ability of users without data or computer science expertise to perform complex image analysis.

TRPV1 controls innate immunity during Citrobacter rodentium enteric infection.

Mucosal immunity is critical to host protection from enteric pathogens and must be carefully controlled to prevent immunopathology. Regulation of immune responses can occur through a diverse range of mechanisms including bi-directional communication with neurons. Among which include specialized sensory neurons that detect noxious stimuli due to the expression of transient receptor potential vanilloid receptor 1 (TRPV1) ion channel and have a significant role in the coordination of host-protective responses to enteric bacterial pathogens. Here we have used the mouse-adapted attaching and effacing pathogen Citrobacter rodentium to assess the specific role of TRPV1 in coordinating the host response. TRPV1 knockout (TRPV1-/-) mice had a significantly higher C. rodentium burden in the distal colon and fecal pellets compared to wild-type (WT) mice. Increased bacterial burden was correlated with significantly increased colonic crypt hyperplasia and proliferating intestinal epithelial cells in TRPV1-/- mice compared to WT. Despite the increased C. rodentium burden and histopathology, the recruitment of colonic T cells producing IFNγ, IL-17, or IL-22 was similar between TRPV1-/- and WT mice. In evaluating the innate immune response, we identified that colonic neutrophil recruitment in C. rodentium infected TRPV1-/- mice was significantly reduced compared to WT mice; however, this was independent of neutrophil development and maturation within the bone marrow compartment. TRPV1-/- mice were found to have significantly decreased expression of the neutrophil-specific chemokine Cxcl6 and the adhesion molecules Icam1 in the distal colon compared to WT mice. Corroborating these findings, a significant reduction in ICAM-1 and VCAM-1, but not MAdCAM-1 protein on the surface of colonic blood endothelial cells from C. rodentium infected TRPV1-/- mice compared to WT was observed. These findings demonstrate the critical role of TRPV1 in regulating the host protective responses to enteric bacterial pathogens, and mucosal immune responses.

TRPV1 controls innate immunity during Citrobacter rodentium enteric infection.

Mucosal immunity is critical to host protection from enteric pathogens and must be carefully controlled to prevent immunopathology. Regulation of immune responses can occur through a diverse range of mechanisms including bi-directional communication with the neurons. Among which include specialized sensory neurons that detect noxious stimuli due to the expression of transient receptor potential vanilloid receptor 1 (TRPV1) ion channel and have a significant role in the coordination of host-protective responses to enteric bacterial pathogens. Here we have used the mouse-adapted attaching and effacing pathogen Citrobacter rodentium to assess the specific role of the TRPV1 channel in coordinating the host response. TRPV1 knockout (TRPV1-/-) mice had a significantly higher C. rodentium burden in the distal colon and fecal pellets compared to wild-type (WT) mice. Increased bacterial burden was correlated with significantly increased colonic crypt hyperplasia and proliferating intestinal epithelial cells in TRPV1-/- mice compared to WT. Despite the increased C. rodentium burden and histopathology, the recruitment of colonic T cells producing IFNγ, IL-17, or IL-22 was similar between TRPV1-/- and WT mice. In evaluating the innate immune response, we identified that colonic neutrophil recruitment in C. rodentium infected TRPV1-/- mice was significantly reduced compared to WT mice; however, this was independent of neutrophil development and maturation within the bone marrow compartment. TRPV1-/- mice were found to have significantly decreased expression of the neutrophil-specific chemokine Cxcl6 and the adhesion molecules Icam1 in the distal colon compared to WT mice. Corroborating these findings, a significant reduction in ICAM-1 and VCAM-1, but not MAdCAM-1 protein on the surface of colonic blood endothelial cells from C. rodentium infected TRPV1-/- mice compared to WT was observed. These findings demonstrate the critical role of TRPV1 in regulating the host protective responses to enteric bacterial pathogens, and mucosal immune responses.

The diversity of neuroimmune circuits controlling lung inflammation.

It is becoming increasingly appreciated that the nervous and immune systems communicate bidirectionally to regulate immunological outcomes in a variety of organs including the lung. Activation of neuronal signaling can be induced by inflammation, tissue damage, or pathogens to evoke or reduce immune cell activation in what has been termed a neuroimmune reflex. In the periphery, these reflexes include the cholinergic anti-inflammatory pathway, sympathetic reflex, and sensory nociceptor-immune cell pathways. Continual advances in neuroimmunology in peripheral organ systems have fueled small-scale clinical trials that have yielded encouraging results for a range of immunopathologies such as rheumatoid arthritis. Despite these successes, several limitations should give clinical investigators pause in the application of neural stimulation as a therapeutic for lung inflammation, especially if inflammation arises from a novel pathogen. In this review, the general mechanisms of each reflex, the evidence for these circuits in the control of lung inflammation, and the key knowledge gaps in our understanding of these neuroimmune circuits will be discussed. These limitations can be overcome not only through a better understanding of neuroanatomy but also through a systematic evaluation of stimulation parameters using immune activation in lung tissues as primary readouts. Our rapidly evolving understanding of the nervous and immune systems highlights the importance of communication between these cells in health and disease. This integrative approach has tremendous potential in the development of targeted therapeutics if specific challenges can be overcome.

Neonatal Enteropathogenic Escherichia coli Infection Disrupts Microbiota-Gut-Brain Axis Signaling.

Diarrheal diseases are a leading cause of death in children under the age of 5 years worldwide. Repeated early-life exposures to diarrheal pathogens can result in comorbidities including stunted growth and cognitive deficits, suggesting an impairment in the microbiota-gut-brain (MGB) axis. Neonatal C57BL/6 mice were infected with enteropathogenic Escherichia coli (EPEC) (strain e2348/69; ΔescV [type III secretion system {T3SS} mutant]) or the vehicle (Luria-Bertani [LB] broth) via orogastric gavage at postnatal day 7 (P7). Behavior (novel-object recognition [NOR] task, light/dark [L/D] box, and open-field test [OFT]), intestinal physiology (Ussing chambers), and the gut microbiota (16S Illumina sequencing) were assessed in adulthood (6 to 8 weeks of age). Neonatal infection of mice with EPEC, but not the T3SS mutant, caused ileal inflammation in neonates and impaired recognition memory (NOR task) in adulthood. Cognitive impairments were coupled with increased neurogenesis (Ki67 and doublecortin immunostaining) and neuroinflammation (increased microglia activation [Iba1]) in adulthood. Intestinal pathophysiology in adult mice was characterized by increased secretory state (short-circuit current [Isc]) and permeability (conductance) (fluorescein isothiocyanate [FITC]-dextran flux) in the ileum and colon of neonatally EPEC-infected mice, along with increased expression of proinflammatory cytokines (Tnfα, Il12, and Il6) and pattern recognition receptors (Nod1/2 and Tlr2/4). Finally, neonatal EPEC infection caused significant dysbiosis of the gut microbiota, including decreased Firmicutes, in adulthood. Together, these findings demonstrate that infection in early life can significantly impair the MGB axis in adulthood.

Divergence of neuroimmune circuits activated by afferent and efferent vagal nerve stimulation in the regulation of inflammation.

KEY POINTS: It has previously been shown that afferent and efferent vagal nerve stimulation potently inhibits lipopolysaccharide (LPS)-induced inflammation Our data show inhibition of inflammation by efferent but not afferent vagal nerve stimulation requires T-cell derived acetylcholine We show that afferent and efferent neuroimmune circuits require ß2 -adrenergic receptor signalling ABSTRACT: Chronic inflammation due to inappropriate immune cell activation can have significant effects on a variety of organ systems, reducing lifespan and quality of life. As such, highly targeted control of immune cell activation is a major therapeutic goal. Vagus nerve stimulation (VNS) has emerged as a therapeutic modality that exploits neuroimmune communication to reduce immune cell activation and consequently inflammation. Although vagal efferent fibres were originally identified as the primary driver of anti-inflammatory actions, the vagus nerve in most species of animals predominantly comprises afferent fibres. Stimulation of vagal afferent fibres can also reduce inflammation; it is, however, uncertain how these two neuroimmune circuits diverge. Here we show that afferent VNS induces a mechanism distinct from efferent VNS, ameliorating lipopolysaccharide (LPS)-induced inflammation independently of T-cell derived acetylcholine (ACh) which is required by efferent VNS. Using a ß2 -adrenergic receptor antagonist (ß2 -AR), we find that immune regulation induced by intact, afferent, or efferent VNS occurs in a ß2- AR-dependent manner. Together, our findings indicate that intact VNS activates at least two distinct neuroimmune circuits each with unique mechanisms of action. Selective targeting of either the vagal efferent or afferent fibres may provide more personalized, robust and effective control over inappropriate immune responses.

Neural Immune Communication in the Control of Host-Bacterial Pathogen Interactions in the Gastrointestinal Tract.

The orchestration of host immune responses to enteric bacterial pathogens is a complex process involving the integration of numerous signals, including from the nervous system. Despite the recent progress in understanding the contribution of neuroimmune interactions in the regulation of inflammation, the mechanisms and effects of this communication during enteric bacterial infection are only beginning to be characterized. As part of this neuroimmune communication, neurons specialized to detect painful or otherwise noxious stimuli can respond to bacterial pathogens. Highlighting the complexity of these systems, the immunological consequences of sensory neuron activation can be either host adaptive or maladaptive, depending on the pathogen and organ system. These are but one of many types of neuroimmune circuits, with the vagus nerve and sympathetic innervation of numerous organs now known to modulate immune cell function and therefore dictate immunological outcomes during health and disease. Here, we review the evidence for neuroimmune communication in response to bacterial pathogens, and then discuss the consequences to host morbidity and mortality during infection of the gastrointestinal tract.

Sensory Nociceptive Neurons Contribute to Host Protection During Enteric Infection With Citrobacter rodentium.

BACKGROUND: Neurons are an integral component of the immune system that functions to coordinate responses to bacterial pathogens. Sensory nociceptive neurons that can detect bacterial pathogens are found throughout the body with dense innervation of the intestinal tract. METHODS: In this study, we assessed the role of these nerves in the coordination of host defenses to Citrobacter rodentium. Selective ablation of nociceptive neurons significantly increased bacterial burden 10 days postinfection and delayed pathogen clearance. RESULTS: Because the sensory neuropeptide CGRP (calcitonin gene-related peptide) regulates host responses during infection of the skin, lung, and small intestine, we assessed the role of CGRP receptor signaling during C rodentium infection. Although CGRP receptor blockade reduced certain proinflammatory gene expression, bacterial burden and Il-22 expression was unaffected. CONCLUSIONS: Our data highlight that sensory nociceptive neurons exert a significant host protective role during C rodentium infection, independent of CGRP receptor signaling.

Perfect timing: circadian rhythms, sleep, and immunity - an NIH workshop summary.

Recent discoveries demonstrate a critical role for circadian rhythms and sleep in immune system homeostasis. Both innate and adaptive immune responses - ranging from leukocyte mobilization, trafficking, and chemotaxis to cytokine release and T cell differentiation -are mediated in a time of day-dependent manner. The National Institutes of Health (NIH) recently sponsored an interdisciplinary workshop, "Sleep Insufficiency, Circadian Misalignment, and the Immune Response," to highlight new research linking sleep and circadian biology to immune function and to identify areas of high translational potential. This Review summarizes topics discussed and highlights immediate opportunities for delineating clinically relevant connections among biological rhythms, sleep, and immune regulation.

Nod-like receptors are critical for gut-brain axis signalling in mice.

KEY POINTS: •Nucleotide binding oligomerization domain (Nod)-like receptors regulate cognition, anxiety and hypothalamic-pituitary-adrenal axis activation. •Nod-like receptors regulate central and peripheral serotonergic biology. •Nod-like receptors are important for maintenance of gastrointestinal physiology. •Intestinal epithelial cell expression of Nod1 receptors regulate behaviour. ABSTRACT: Gut-brain axis signalling is critical for maintaining health and homeostasis. Stressful life events can impact gut-brain signalling, leading to altered mood, cognition and intestinal dysfunction. In the present study, we identified nucleotide binding oligomerization domain (Nod)-like receptors (NLR), Nod1 and Nod2, as novel regulators for gut-brain signalling. NLR are innate immune pattern recognition receptors expressed in the gut and brain, and are important in the regulation of gastrointestinal physiology. We found that mice deficient in both Nod1 and Nod2 (NodDKO) demonstrate signs of stress-induced anxiety, cognitive impairment and depression in the context of a hyperactive hypothalamic-pituitary-adrenal axis. These deficits were coupled with impairments in the serotonergic pathway in the brain, decreased hippocampal cell proliferation and immature neurons, as well as reduced neural activation. In addition, NodDKO mice had increased gastrointestinal permeability and altered serotonin signalling in the gut following exposure to acute stress. Administration of the selective serotonin reuptake inhibitor, fluoxetine, abrogated behavioural impairments and restored serotonin signalling. We also identified that intestinal epithelial cell-specific deletion of Nod1 (VilCre+ Nod1f/f ), but not Nod2, increased susceptibility to stress-induced anxiety-like behaviour and cognitive impairment following exposure to stress. Together, these data suggest that intestinal epithelial NLR are novel modulators of gut-brain communication and may serve as potential novel therapeutic targets for the treatment of gut-brain disorders.

Functional circuitry of neuro-immune communication in the mesenteric lymph node and spleen.

The peripheral nervous system is an active participant in immune responses capable of blocking aberrant activation of a variety of immune cells. As one of these neuro-immune circuits, the cholinergic anti-inflammatory pathway has been well established to reduce the severity of several immunopathologies. While the activation of this pathway by vagal nerve stimulation requires sympathetic innervation of the spleen, the neuro-immune circuitry remains highly controversial. Neuro-immune pathways in other lymphoid tissues such as mesenteric lymph nodes (MLN) that are critical to the surveillance of the small intestine and proximal colon have not been assessed. Using conditionally expressed Channelrhodopsin, selective stimulation of sympathetic post-ganglionic neurons in the superior mesenteric ganglion (SMG) prevented macrophage activation and LPS-induced TNFα production in the spleen and MLN, but not in the inguinal LN. Site selective stimulation of the SMG induced the release of norepinephrine, resulting in ß2AR dependent acetylcholine release in the MLN and spleen. VNS-evoked release of norepinephrine and acetylcholine in the MLN and spleen was significantly reduced using selective optogenetic blockade applied at the SMG. Additionally, this optogenetic blockade restored LPS-induced TNFα production, despite VNS. These studies identify the superior mesenteric ganglion as a critical node in a neuro-immune circuit that can inhibit immune function in the MLN and the spleen.

T-cell derived acetylcholine aids host defenses during enteric bacterial infection with Citrobacter rodentium.

The regulation of mucosal immune function is critical to host protection from enteric pathogens but is incompletely understood. The nervous system and the neurotransmitter acetylcholine play an integral part in host defense against enteric bacterial pathogens. Here we report that acetylcholine producing-T-cells, as a non-neuronal source of ACh, were recruited to the colon during infection with the mouse pathogen Citrobacter rodentium. These ChAT+ T-cells did not exclusively belong to one Th subset and were able to produce IFNγ, IL-17A and IL-22. To interrogate the possible protective effect of acetylcholine released from these cells during enteric infection, T-cells were rendered deficient in their ability to produce acetylcholine through a conditional gene knockout approach. Significantly increased C. rodentium burden was observed in the colon from conditional KO (cKO) compared to WT mice at 10 days post-infection. This increased bacterial burden in cKO mice was associated with increased expression of the cytokines IL-1ß, IL-6, and TNFα, but without significant changes in T-cell and ILC associated IL-17A, IL-22, and IFNγ, or epithelial expression of antimicrobial peptides, compared to WT mice. Despite the increased expression of pro-inflammatory cytokines during C. rodentium infection, inducible nitric oxide synthase (Nos2) expression was significantly reduced in intestinal epithelial cells of ChAT T-cell cKO mice 10 days post-infection. Additionally, a cholinergic agonist enhanced IFNγ-induced Nos2 expression in intestinal epithelial cell in vitro. These findings demonstrated that acetylcholine, produced by specialized T-cells that are recruited during C. rodentium infection, are a key mediator in host-microbe interactions and mucosal defenses.

Adenylyl Cyclase 6 Mediates Inhibition of TNF in the Inflammatory Reflex.

Macrophage cytokine production is regulated by neural signals, for example in the inflammatory reflex. Signals in the vagus and splenic nerves are relayed by choline acetyltransferase+ T cells that release acetylcholine, the cognate ligand for alpha7 nicotinic acetylcholine subunit-containing receptors (α7nAChR), and suppress TNF release in macrophages. Here, we observed that electrical vagus nerve stimulation with a duration of 0.1-60 s significantly reduced systemic TNF release in experimental endotoxemia. This suppression of TNF was sustained for more than 24 h, but abolished in mice deficient in the α7nAChR subunit. Exposure of primary human macrophages and murine RAW 264.7 macrophage-like cells to selective ligands for α7nAChR for 1 h in vitro attenuated TNF production for up to 24 h in response to endotoxin. Pharmacological inhibition of adenylyl cyclase (AC) and knockdown of adenylyl cyclase 6 (AC6) or c-FOS abolished cholinergic suppression of endotoxin-induced TNF release. These findings indicate that action potentials in the inflammatory reflex trigger a change in macrophage behavior that requires AC and phosphorylation of the cAMP response element binding protein (CREB). These observations further our mechanistic understanding of neural regulation of inflammation and may have implications for development of bioelectronic medicine treatment of inflammatory diseases.

Neuroimmune Communication in Health and Disease.

The immune and nervous systems are tightly integrated, with each system capable of influencing the other to respond to infectious or inflammatory perturbations of homeostasis. Recent studies demonstrating the ability of neural stimulation to significantly reduce the severity of immunopathology and consequently reduce mortality have led to a resurgence in the field of neuroimmunology. Highlighting the tight integration of the nervous and immune systems, afferent neurons can be activated by a diverse range of substances from bacterial-derived products to cytokines released by host cells. While activation of vagal afferents by these substances dominates the literature, additional sensory neurons are responsive as well. It is becoming increasingly clear that although the cholinergic anti-inflammatory pathway has become the predominant model, a multitude of functional circuits exist through which neuronal messengers can influence immunological outcomes. These include pathways whereby efferent signaling occurs independent of the vagus nerve through sympathetic neurons. To receive input from the nervous system, immune cells including B and T cells, macrophages, and professional antigen presenting cells express specific neurotransmitter receptors that affect immune cell function. Specialized immune cell populations not only express neurotransmitter receptors, but express the enzymatic machinery required to produce neurotransmitters, such as acetylcholine, allowing them to act as signaling intermediaries. Although elegant experiments have begun to decipher some of these interactions, integration of these molecules, cells, and anatomy into defined neuroimmune circuits in health and disease is in its infancy. This review describes these circuits and highlights continued challenges and opportunities for the field.

Neuroanatomy of the spleen: Mapping the relationship between sympathetic neurons and lymphocytes.

The nervous system plays a profound regulatory role in maintaining appropriate immune responses by signaling to immune cells. These immune cells, including B- and T-cells, can further act as intermediary messengers, with subsets of B- and T-cells expressing choline acetyltransferase (ChAT), the enzyme required for acetylcholine (ACh) synthesis. Neural control of ACh release from ChAT+ T-cells can have powerful immune implications, regulating lymphocyte trafficking, inflammation, and prevent death due to experimental septic shock. Although ACh release from T-cells has been proposed to occur following norepinephrine (NE) released from sympathetic nerve terminals in the spleen, it is unknown how this communication occurs. While it was proposed that tyrosine hydroxylase (TH+) axons form synapse-like structures with ChAT+ T-cells, there is scant evidence to support or refute this phenomenon. With this in mind, we sought to determine the relative abundance of ChAT+ B- and T-cells in close proximity to TH+ axons, and determine what factors contribute to their localization in the spleen. Using confocal microscopy of tissue sections and three-dimensional imaging of intact spleen, we confirmed that ChAT+ B-cells exceed the number of ChAT+ T-cells, and overall few ChAT+ B- or T-cells are located close to TH+ fibers compared to total numbers. The organized location of ChAT+ lymphocytes within the spleen suggested that these cells were recruited by chemokine gradients. We identified ChAT+ B- and T-cells express the chemokine receptor CXCR5; indicating that these cells can respond to CXCL13 produced by stromal cells expressing the ß2 adrenergic receptor in the spleen. Our findings suggest that sympathetic innervation contributes to organization of ChAT+ immune cells in the white pulp of the spleen by regulating CXCL13. Supporting this contention, chemical sympathectomy significantly reduced expression of this chemokine. Together, we demonstrated that there does not appear to be a basis for synaptic neuro-immune communication, and that sympathetic innervation can modulate immune function through altering stromal cell chemokine production.

The IgM receptor FcµR limits tonic BCR signaling by regulating expression of the IgM BCR.

The FcµR receptor for the crystallizable fragment (Fc) of immunoglobulin M (IgM) can function as a cell-surface receptor for secreted IgM on a variety of cell types. We found here that FcµR was also expressed in the trans-Golgi network of developing B cells, where it constrained transport of the IgM-isotype BCR (IgM-BCR) but not of the IgD-isotype BCR (IgD-BCR). In the absence of FcµR, the surface expression of IgM-BCR was increased, which resulted in enhanced tonic BCR signaling. B-cell-specific deficiency in FcµR enhanced the spontaneous differentiation of B-1 cells, which resulted in increased serum concentrations of natural IgM and dysregulated homeostasis of B-2 cells; this caused the spontaneous formation of germinal centers, increased titers of serum autoantibodies and excessive accumulation of B cells. Thus, FcµR serves as a critical regulator of B cell biology by constraining the transport and cell-surface expression of IgM-BCR.

Neuro-immune interactions in the cholinergic anti-inflammatory reflex.

Communication between the nervous and immune systems can significantly alter immune cell function in a number of inflammatory diseases. Elegant studies have defined a basic functional circuit in a "cholinergic anti-inflammatory pathway" that highlights a unique role for the vagus nerve, and has brought about a resurgence in the field of neuro-immunology. This research has further identified that in addition to tonic signals that can restrain immune cell activation; the anti-inflammatory reflex arc is amiable to targeted stimulation as a therapeutic modality. The success of vagal electrical neural stimulation in a plethora of pre-clinical inflammation models has spurred the development of "electroceuticals" or neurostimulatory devices in the treatment of chronic inflammation. This development has begun despite addressing of fundamental questions such as the functional neural circuitry being crudely mapped and unresolved mechanisms of action of acetylcholine on target immune cells. Perhaps fortuitously, rapid advances in neuroscience techniques may allow us to begin to answer some of these longstanding questions and clarify recent controversies.

A Convenient Method for Evaluating Epithelial Cell Proliferation in the Whole Mammary Glands of Female Mice.

The mammary glands (MG) undergo rapid expansion of the ductal network during puberty in response to endocrine cues including the potent mitogenic effects of estrogen. The proliferation of mammary epithelial cells occurs in a spatially distinctive manner, where terminal end buds located at the ductal termini are the primary site of cell division. Here, we present a relatively high throughput approach to spatially assess epithelial cell proliferation in whole mouse MG using histochemical detection of 5-ethynyl-2'-deoxyuridine in conjunction with a standard curve-based data deconvolution technique to semiquantitatively measure proliferation via wide-field epifluorescent microscopy. This approach was validated against the "gold standard" of counting labeled nuclei from confocal images utilizing computer-assisted image analysis. Our method proved sensitive enough to describe the significant and spatially variable proliferative response to low-dose estrogen after 108 hours. This flexible method presents a timely and economical approach to obtaining spatial information regarding epithelial cell proliferation in the mouse MG.