Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

Therapeutic Treatment of Superoxide Dismutase 1 (G93A) Amyotrophic Lateral Sclerosis Model Mice with Medical Ozone Decelerates Trigeminal Motor Neuron Degeneration, Attenuates Microglial Proliferation, and Preserves Monocyte Levels in Mesenteric Lymph Nodes.

Amyotrophic lateral sclerosis (ALS) is an incurable and lethal neurodegenerative disease in which progressive motor neuron loss and associated inflammation represent major pathology hallmarks. Both the prevention of neuronal loss and neuro-destructive inflammation are still unmet challenges. Medical ozone, an ozonized oxygen mixture (O3/O2), has been shown to elicit profound immunomodulatory effects in peripheral organs, and beneficial effects in the aging brain. We investigated, in a preclinical drug testing approach, the therapeutic potential of a five-day O3/O2i.p. treatment regime at the beginning of the symptomatic disease phase in the superoxide dismutase (SOD1G93A) ALS mouse model. Clinical assessment of SOD1G93A mice revealed no benefit of medical ozone treatment over sham with respect to gross body weight, motor performance, disease duration, or survival. In the brainstem of end stage SOD1G93A mice, however, neurodegeneration was found decelerated, and SOD1-related vacuolization was reduced in the motor trigeminal nucleus in the O3/O2 treatment group when compared to sham-treated mice. In addition, microglia proliferation was less pronounced in the brainstem, while the hypertrophy of astroglia remained largely unaffected. Finally, monocyte numbers were reduced in the blood, spleen, and mesenteric lymph nodes at postnatal day 60 in SOD1G93A mice. A further decrease in monocyte numbers seen in mesenteric lymph nodes from sham-treated SOD1G93A mice at an advanced disease stage, however, was prevented by medical ozone treatment. Collectively, our study revealed a select neuroprotective and possibly anti-inflammatory capacity for medical ozone when applied as a therapeutic agent in SOD1G93A ALS mice.

Development of epithelial cholinergic chemosensory cells of the urethra and trachea of mice.

Cholinergic chemosensory cells (CCC) are infrequent epithelial cells with immunosensor function, positioned in mucosal epithelia preferentially near body entry sites in mammals including man. Given their adaptive capacity in response to infection and their role in combatting pathogens, we here addressed the time points of their initial emergence as well as their postnatal development from first exposure to environmental microbiota (i.e., birth) to adulthood in urethra and trachea, utilizing choline acetyltransferase (ChAT)-eGFP reporter mice, mice with genetic deletion of MyD88, toll-like receptor-2 (TLR2), TLR4, TLR2/TLR4, and germ-free mice. Appearance of CCC differs between the investigated organs. CCC of the trachea emerge during embryonic development at E18 and expand further after birth. Urethral CCC show gender diversity and appear first at P6-P10 in male and at P11-P20 in female mice. Urethrae and tracheae of MyD88- and TLR-deficient mice showed significantly fewer CCC in all four investigated deficient strains, with the effect being most prominent in the urethra. In germ-free mice, however, CCC numbers were not reduced, indicating that TLR2/4-MyD88 signaling, but not vita-PAMPs, governs CCC development. Collectively, our data show a marked postnatal expansion of CCC populations with distinct organ-specific features, including the relative impact of TLR2/4-MyD88 signaling. Strong dependency on this pathway (urethra) correlates with absence of CCC at birth and gender-specific initial development and expansion dynamics, whereas moderate dependency (trachea) coincides with presence of first CCC at E18 and sex-independent further development.

Disruption of calcitonin gene-related peptide signaling accelerates muscle denervation and dampens cytotoxic neuroinflammation in SOD1 mutant mice.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Neuronal vacuolization and glial activation are pathologic hallmarks in the superoxide dismutase 1 (SOD1) mouse model of ALS. Previously, we found the neuropeptide calcitonin gene-related peptide (CGRP) associated with vacuolization and astrogliosis in the spinal cord of these mice. We now show that CGRP abundance positively correlated with the severity of astrogliosis, but not vacuolization, in several motor and non-motor areas throughout the brain. SOD1 mice harboring a genetic depletion of the ßCGRP isoform showed reduced CGRP immunoreactivity associated with vacuolization, while motor functions, body weight, survival, and astrogliosis were not altered. When CGRP signaling was completely disrupted through genetic depletion of the CGRP receptor component, receptor activity-modifying protein 1 (RAMP1), hind limb muscle denervation, and loss of muscle performance were accelerated, while body weight and survival were not affected. Dampened neuroinflammation, i.e., reduced levels of astrogliosis in the brain stem already in the pre-symptomatic disease stage, and reduced microgliosis and lymphocyte infiltrations during the late disease phase were additional neuropathology features in these mice. On the molecular level, mRNA expression levels of brain-derived neurotrophic factor (BDNF) and those of the anti-inflammatory cytokine interleukin 6 (IL-6) were elevated, while those of several pro-inflammatory cytokines found reduced in the brain stem of RAMP1-deficient SOD1 mice at disease end stage. Our results thus identify an important, possibly dual role of CGRP in ALS pathogenesis.

Bitter triggers acetylcholine release from polymodal urethral chemosensory cells and bladder reflexes.

Chemosensory cells in the mucosal surface of the respiratory tract ("brush cells") use the canonical taste transduction cascade to detect potentially hazardous content and trigger local protective and aversive respiratory reflexes on stimulation. So far, the urogenital tract has been considered to lack this cell type. Here we report the presence of a previously unidentified cholinergic, polymodal chemosensory cell in the mammalian urethra, the potential portal of entry for bacteria and harmful substances into the urogenital system, but not in further centrally located parts of the urinary tract, such as the bladder, ureter, and renal pelvis. Urethral brush cells express bitter and umami taste receptors and downstream components of the taste transduction cascade; respond to stimulation with bitter (denatonium), umami (monosodium glutamate), and uropathogenic Escherichia coli; and release acetylcholine to communicate with other cells. They are approached by sensory nerve fibers expressing nicotinic acetylcholine receptors, and intraurethral application of denatonium reflexively increases activity of the bladder detrusor muscle in anesthetized rats. We propose a concept of urinary bladder control involving a previously unidentified cholinergic chemosensory cell monitoring the chemical composition of the urethral luminal microenvironment for potential hazardous content.

Satb2-independent acquisition of the cholinergic sudomotor phenotype in rodents.

Expression of Satb2 (Special AT-rich sequence-binding protein-2) elicits expression of the vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) in cultured rat sympathetic neurons exposed to soluble differentiation factors. Here, we determined whether or not Satb2 plays a similar role in cholinergic differentiation in vivo, by comparing the postnatal profile of Satb2 expression in the rodent stellate ganglion to that of VAChT and ChAT. Throughout postnatal development, VAChT and ChAT were found to be co-expressed in a numerically stable subpopulation of rat stellate ganglion neurons. Nerve fibers innervating rat forepaw sweat glands on P1 were VAChT immunoreactive, while ChAT was detectable at this target only after P5. The postnatal abundance of VAChT transcripts in the stellate ganglion was at maximum already on P1, whereas ChAT mRNA levels increased from low levels on P1 to reach maximum levels between P5 and P21. Satb2 mRNA was detected in cholinergic neurons in the stellate ganglion beginning with P8, thus coincident with the onset of unequivocal detection of ChAT immunoreactivity in forepaw sweat gland endings. Satb2 knockout mice exhibited no change in the P1 cholinergic VAChT/ChAT co-phenotype in stellate ganglion neurons. Thus, cholinergic phenotype maturation involves first, early target (sweat-gland)-independent expression and trafficking of VAChT, and later, potentially target- and Satb2-dependent elevation of ChAT mRNA and protein transport into sweat gland endings. In rat sudomotor neurons that, unlike mouse sudomotor neurons, co-express calcitonin gene-related peptide (CGRP), Satb2 may also be related to the establishment of species-specific neuropeptide co-phenotypes during postnatal development.

Elevated fecal calprotectin in patients with Alzheimer's dementia indicates leaky gut.

Fecal concentrations of calprotectin were examined in 22 patients with Alzheimer's disease (AD) and compared with serum concentrations of aromatic amino acids. Calprotectin concentrations were mean ± SEM 140 ± 31.9 mg/kg, 16 patients (73%) presented with concentrations outside normal (>50 mg/kg). Concentrations correlated inversely with serum levels of tryptophan, tyrosine and phenylalanine (all p < 0.05). Increased concentrations of fecal calprotectin indicate a disturbed intestinal barrier function in AD patients which could be of relevance for the lowering of essential aromatic amino acids concentrations in the blood.

Cholinergic epithelial cell with chemosensory traits in murine thymic medulla.

Specialized epithelial cells with a tuft of apical microvilli ("brush cells") sense luminal content and initiate protective reflexes in response to potentially harmful substances. They utilize the canonical taste transduction cascade to detect "bitter" substances such as bacterial quorum-sensing molecules. In the respiratory tract, most of these cells are cholinergic and are approached by cholinoceptive sensory nerve fibers. Utilizing two different reporter mouse strains for the expression of choline acetyltransferase (ChAT), we observed intense labeling of a subset of thymic medullary cells. ChAT expression was confirmed by in situ hybridization. These cells showed expression of villin, a brush cell marker protein, and ultrastructurally exhibited lateral microvilli. They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cß2. Reverse transcription and polymerase chain reaction confirmed the expression of Gα-gustducin, TRPM5, and phospholipase Cß2. Thymic "cholinergic chemosensory cells" were often in direct contact with medullary epithelial cells expressing the nicotinic acetylcholine receptor subunit α3. These cells have recently been identified as terminally differentiated epithelial cells (Hassall's corpuscle-like structures in mice). Contacts with nerve fibers (identified by PGP9.5 and CGRP antibodies), however, were not observed. Our data identify, in the thymus, a previously unrecognized presumptive chemosensitive cell that probably utilizes acetylcholine for paracrine signaling. This cell might participate in intrathymic infection-sensing mechanisms.

Cholinergic chemosensory cells in the trachea regulate breathing.

In the epithelium of the lower airways, a cell type of unknown function has been termed "brush cell" because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signaling molecules (α-gustducin, phospholipase C(ß2)) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.

Bitter tastants relax the mouse gallbladder smooth muscle independent of signaling through tuft cells and bitter taste receptors.

Disorders of gallbladder motility can lead to serious pathology. Bitter tastants acting upon bitter taste receptors (TAS2R family) have been proposed as a novel class of smooth muscle relaxants to combat excessive contraction in the airways and other organs. To explore whether this might also emerge as an option for gallbladder diseases, we here tested bitter tastants for relaxant properties and profiled Tas2r expression in the mouse gallbladder. In organ bath experiments, the bitter tastants denatonium, quinine, dextromethorphan, and noscapine, dose-dependently relaxed the pre-contracted gallbladder. Utilizing gene-deficient mouse strains, neither transient receptor potential family member 5 (TRPM5), nor the Tas2r143/Tas2r135/Tas2r126 gene cluster, nor tuft cells proved to be required for this relaxation, indicating direct action upon smooth muscle cells (SMC). Accordingly, denatonium, quinine and dextromethorphan increased intracellular calcium concentration preferentially in isolated gallbladder SMC and, again, this effect was independent of TRPM5. RT-PCR revealed transcripts of Tas2r108, Tas2r126, Tas2r135, Tas2r137, and Tas2r143, and analysis of gallbladders from mice lacking tuft cells revealed preferential expression of Tas2r108 and Tas2r137 in tuft cells. A TAS2R143-mCherry reporter mouse labeled tuft cells in the gallbladder epithelium. An in silico analysis of a scRNA sequencing data set revealed Tas2r expression in only few cells of different identity, and from in situ hybridization histochemistry, which did not label distinct cells. Our findings demonstrate profound tuft cell- and TRPM5-independent relaxing effects of bitter tastants on gallbladder smooth muscle, but do not support the concept that these effects are mediated by bitter receptors.

Selective mitochondrial Ca2+ uptake deficit in disease endstage vulnerable motoneurons of the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that targets some somatic motoneuron populations, while others, e.g. those of the oculomotor system, are spared. The pathophysiological basis of this pattern of differential vulnerability, which is preserved in a transgenic mouse model of amyotrophic lateral sclerosis (SOD1(G93A)), and the mechanism of neurodegeneration in general are unknown. Hyperexcitability and calcium dysregulation have been proposed by others on the basis of data from juvenile mice that are, however, asymptomatic. No studies have been done with symptomatic mice following disease progression to the disease endstage. Here, we developed a new brainstem slice preparation for whole-cell patch-clamp recordings and single cell fura-2 calcium imaging to study motoneurons in adult wild-type and SOD1(G93A) mice up to disease endstage. We analysed disease-stage-dependent electrophysiological properties and intracellular Ca(2+) handling of vulnerable hypoglossal motoneurons in comparison to resistant oculomotor neurons. Thereby, we identified a transient hyperexcitability in presymptomatic but not in endstage vulnerable motoneurons. Additionally, we revealed a remodelling of intracellular Ca(2+) clearance within vulnerable but not resistant motoneurons at disease endstage characterised by a reduction of uniporter-dependent mitochondrial Ca(2+) uptake and enhanced Ca(2+) extrusion across the plasma membrane. Our study challenged the notion that hyperexcitability is a direct cause of neurodegeneration in SOD1(G93A) mice, but molecularly identified a Ca(2+) clearance deficit in motoneurons and an adaptive Ca(2+) handling strategy that might be targeted by future therapeutic strategies.

PACAP signaling exerts opposing effects on neuroprotection and neuroinflammation during disease progression in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic peptide with autocrine neuroprotective and paracrine anti-inflammatory properties in various models of acute neuronal damage and neurodegenerative diseases. Therefore, we examined a possible beneficial role of endogenous PACAP in the superoxide dismutase 1, SOD1(G93A), mouse model of amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disease particularly affecting somatomotor neurons. In wild-type mice, somatomotor and visceromotor neurons in brain stem and spinal cord were found to express the PACAP specific receptor PAC1, but only visceromotor neurons expressed PACAP as a potential autocrine source of regulation of these receptors. In SOD1(G93A) mice, only a small subset of the surviving somatomotor neurons showed induction of PACAP mRNA, and somatomotor neuron degeneration was unchanged in PACAP-deficient SOD1(G93A) mice. Pre-ganglionic sympathetic visceromotor neurons were found to be resistant in SOD1(G93A) mice, while pre-ganglionic parasympathetic neurons degenerated during ALS disease progression in this mouse model. PACAP-deficient SOD1(G93A) mice showed even greater pre-ganglionic parasympathetic neuron loss compared to SOD1(G93A) mice, and additional degeneration of pre-ganglionic sympathetic neurons. Thus, constitutive expression of PACAP and PAC1 may confer neuroprotection to central visceromotor neurons in SOD1(G93A) mice via autocrine pathways. Regarding the progression of neuroinflammation, the switch from amoeboid to hypertrophic microglial phenotype observed in SOD1(G93A) mice was absent in PACAP-deficient SOD1(G93A) mice. Thus, endogenous PACAP may promote microglial cytodestructive functions thought to drive ALS disease progression. This hypothesis was consistent with prolongation of life expectancy and preserved tongue motor function in PACAP-deficient SOD1(G93A) mice, compared to SOD1(G93A) mice. Given the protective role of PACAP expression in visceromotor neurons and the opposing effect on microglial function in SOD1(G93A) mice, both PACAP agonism and antagonism may be promising therapeutic tools for ALS treatment, if stage of disease progression and targeting the specific auto- and paracrine signaling pathways are carefully considered.

The Impact of Gut Microbiota-Derived Metabolites on the Tumor Immune Microenvironment.

Prevention of the effectiveness of anti-tumor immune responses is one of the canonical cancer hallmarks. The competition for crucial nutrients within the tumor microenvironment (TME) between cancer cells and immune cells creates a complex interplay characterized by metabolic deprivation. Extensive efforts have recently been made to understand better the dynamic interactions between cancer cells and surrounding immune cells. Paradoxically, both cancer cells and activated T cells are metabolically dependent on glycolysis, even in the presence of oxygen, a metabolic process known as the Warburg effect. The intestinal microbial community delivers various types of small molecules that can potentially augment the functional capabilities of the host immune system. Currently, several studies are trying to explore the complex functional relationship between the metabolites secreted by the human microbiome and anti-tumor immunity. Recently, it has been shown that a diverse array of commensal bacteria synthetizes bioactive molecules that enhance the efficacy of cancer immunotherapy, including immune checkpoint inhibitor (ICI) treatment and adoptive cell therapy with chimeric antigen receptor (CAR) T cells. In this review, we highlight the importance of commensal bacteria, particularly of the gut microbiota-derived metabolites that are capable of shaping metabolic, transcriptional and epigenetic processes within the TME in a therapeutically meaningful way.

A ketogenic diet substantially reshapes the human metabolome.

BACKGROUND: Western dietary habits (WD) have been shown to promote chronic inflammation, which favors the development of many of today's non-communicable diseases. Recently, ketogenic diets (KD) have emerged as an immune-regulating countermeasure for WD-induced metaflammation. To date, beneficial effects of KD have been solely attributed to the production and metabolism of ketone bodies. Given the drastic change in nutrient composition during KD, it is reasonable to assume that there are widespread changes in the human metabolome also contributing to the impact of KD on human immunity. The current study was conducted to gain insight into the changes of the human metabolic fingerprint associated with KD. This could allow to identify metabolites that may contribute to the overall positive effects on human immunity, but also help to recognize potential health risks of KD. METHODS: We conducted a prospective nutritional intervention study enrolling 40 healthy volunteers to perform a three-week ad-libitum KD. Prior to the start and at the end of the nutritional intervention serum metabolites were quantified, untargeted mass spectrometric metabolome analyses and urine analyses of the tryptophan pathway were performed. RESULTS: KD led to a marked reduction of insulin (-21.45% ± 6.44%, p = 0.0038) and c-peptide levels (-19.29% ± 5.45%, p = 0.0002) without compromising fasting blood glucose. Serum triglyceride concentration decreased accordingly (-13.67% ± 5.77%, p = 0.0247), whereas cholesterol parameters remained unchanged. LC-MS/MS-based untargeted metabolomic analyses revealed a profound shift of the human metabolism towards mitochondrial fatty acid oxidation, comprising highly elevated levels of free fatty acids and acylcarnitines. The serum amino acid (AA) composition was rearranged with lower abundance of glucogenic AA and an increase of BCAA. Furthermore, an increase of anti-inflammatory fatty acids eicosatetraenoic acid (p < 0.0001) and docosahexaenoic acid (p = 0.0002) was detected. Urine analyses confirmed higher utilization of carnitines, indicated by lower carnitine excretion (-62.61% ± 18.11%, p = 0.0047) and revealed changes to the tryptophan pathway depicting reduced quinolinic acid (-13.46% ± 6.12%, p = 0.0478) and elevated kynurenic acid concentrations (+10.70% ± 4.25%, p = 0.0269). CONCLUSIONS: A KD fundamentally changes the human metabolome even after a short period of only three weeks. Besides a rapid metabolic switch to ketone body production and utilization, improved insulin and triglyceride levels and an increase in metabolites that mediate anti-inflammation and mitochondrial protection occurred. Importantly, no metabolic risk factors were identified. Thus, a ketogenic diet could be considered as a safe preventive and therapeutic immunometabolic tool in modern medicine. TRIAL REGISTRATION: German Clinical Trials Register; DRKS-ID: DRKS00027992 (www.drks.de).

A succinate/SUCNR1-brush cell defense program in the tracheal epithelium.

Host-derived succinate accumulates in the airways during bacterial infection. Here, we show that luminal succinate activates murine tracheal brush (tuft) cells through a signaling cascade involving the succinate receptor 1 (SUCNR1), phospholipase Cß2, and the cation channel transient receptor potential channel subfamily M member 5 (TRPM5). Stimulated brush cells then trigger a long-range Ca2+ wave spreading radially over the tracheal epithelium through a sequential signaling process. First, brush cells release acetylcholine, which excites nearby cells via muscarinic acetylcholine receptors. From there, the Ca2+ wave propagates through gap junction signaling, reaching also distant ciliated and secretory cells. These effector cells translate activation into enhanced ciliary activity and Cl- secretion, which are synergistic in boosting mucociliary clearance, the major innate defense mechanism of the airways. Our data establish tracheal brush cells as a central hub in triggering a global epithelial defense program in response to a danger-associated metabolite.

Calcitonin gene-related peptide expression levels predict motor neuron vulnerability in the superoxide dismutase 1-G93A mouse model of amyotrophic lateral sclerosis.

In amyotrophic lateral sclerosis (ALS) some motor neurons degenerate while others survive. The molecular mechanisms underlying this selective vulnerability and resistance, respectively, are poorly understood. Since the neuropeptide, calcitonin gene-related peptide (CGRP), is expressed by many but not all motor neurons, we asked if motor neuron CGRP levels predict their vulnerability in the SOD1-G93A mouse model of ALS. In wild type mice three types of somatic motor neurons were distinguished based on their CGRP expression pattern, i.e. highCGRP, lowCGRP, and nonCGRP. Since motor nuclei III, IV and VI contained mostly nonCGRP motor neurons, they defined the oculomotor group. In comparison, the facial group (nuclei V, VII and XII) contained equal numbers of all three types, while the spinomedullary group (ambiguus nucleus and lumbar spinal cord) contained mainly highCGRP motor neurons. Analysis on the transcript level, and of mice lacking the αCGRP isoform, revealed that these group differences in CGRP expression were predominantly based on αCGRP. At disease end-stage in SOD1-G93A mice, group-specific extent of motor neuron loss correlated with CGRP expression as neurons with highCGRP were reduced by 80%, those with lowCGRP by 50%, and nonCGRP motor neurons were not significantly affected in all three groups. Finally, highCGRP motor neuron degeneration preceded lowCGRP motor neuron degeneration during disease progression. Our analysis revealed that the relative abundance of CGRP mRNA and immunoreactivity in motor neurons predicts their vulnerability. CGRP may be an autocrine or paracrine factor promoting motor neuron degeneration in this ALS model.

Transcription factor c-Rel mediates communication between commensal bacteria and mucosal lymphocytes.

The NF-κB transcription factor c-Rel plays a crucial role in promoting and regulating immune responses and inflammation. However, the function of c-Rel in modulating the mucosal immune system is poorly understood. T follicular helper (Tfh) cells and IgA production in gut-associated lymphoid tissues (GALT) such as Peyer's patches (PPs) are important for maintaining the intestinal homeostasis. Here, c-Rel was identified as an essential factor regulating intestinal IgA generation and function of Tfh cells. Genetic deletion of c-Rel resulted in the aberrant formation of germinal centers (GCs) in PPs, significantly reduced IgA generation and defective Tfh cell differentiation. Supporting these findings, the Ag-specific IgA response to Citrobacter rodentium was strongly impaired in c-Rel-deficient mice. Interestingly, an excessive expansion of segmented filamentous bacteria (SFB) was observed in the small intestine of animals lacking c-Rel. Yet, the production of IL-17A, IgA, and IL-21, which are induced by SFB, was impaired due to the lack of transcriptional control by c-Rel. Collectively, the transcriptional activity of c-Rel regulates Tfh cell function and IgA production in the gut, thus preserving the intestinal homeostasis.

Cysteinyl leukotrienes and acetylcholine are biliary tuft cell cotransmitters.

The gallbladder stores bile between meals and empties into the duodenum upon demand and is thereby exposed to the intestinal microbiome. This exposure raises the need for antimicrobial factors, among them, mucins produced by cholangiocytes, the dominant epithelial cell type in the gallbladder. The role of the much less frequent biliary tuft cells is still unknown. We here show that propionate, a major metabolite of intestinal bacteria, activates tuft cells via the short-chain free fatty acid receptor 2 and downstream signaling involving the cation channel transient receptor potential cation channel subfamily M member 5. This results in corelease of acetylcholine and cysteinyl leukotrienes from tuft cells and evokes synergistic paracrine effects upon the epithelium and the gallbladder smooth muscle, respectively. Acetylcholine triggers mucin release from cholangiocytes, an epithelial defense mechanism, through the muscarinic acetylcholine receptor M3. Cysteinyl leukotrienes cause gallbladder contraction through their cognate receptor CysLTR1, prompting emptying and closing. Our results establish gallbladder tuft cells as sensors of the microbial metabolite propionate, initiating dichotomous innate defense mechanisms through simultaneous release of acetylcholine and cysteinyl leukotrienes.

PMA-zeolite can modulate inflammation associated markers in irritable bowel disease - an explorative randomized, double blinded, controlled pilot trial.

OBJECTIVES: Preclinical and clinical data suggest, that the microporous mineral with large inner surface and ion exchanger capability PMA-(Panaceo-micro-activation)-zeolite can bind irritating and inflammation associated chime-constituents. We hypothesised whether or not it can ameliorate subclinical inflammation, and investigated the potential in the management of patients with Irritable Bowel syndrome (IBS). METHODS: The trial design was prospective, randomized, controlled, double-blinded, pilot study with 41 patients. They received orally 3 g of the medicinal product PMA-zeolite or microcrystalline cellulose (control) twice a day. At baseline and after three months the symptom load, blood and stool parameters, like high sensitivity C-reactive protein (hsCRP), zonulin, α1-antitrypsin, interleukin IL-10, and changes in the gut microbiome were determined by means of ANOVA, ANCOVA and non parametric statistical analyses. RESULTS: The IBS-associated symptom scores decreased significantly in both groups (p=0,001) indicating a strong placebo effect. In the verum but not in the placebo group various inflammation related laboratory parameters decreased. The gross statistical comparison revealed a reduction of α1-antitrypsin significant (p=0,037), a lowered inflammation marker hsCRP, paralleled specific microbiome changes (Lactobacillus, Bifidobacteria, and Firmicutes). CONCLUSIONS: The decrease of blood hsCRP and decreased stool α1-antitrypsin suggest that PMA-zeolite possibly can lower inflammation in the gut of IBS patients. The corresponding increase of the immune modulating species Bifidobacteria and Lactobacillus and the reduction of Firmicutes also point at an inflammation ameliorating effect and a possible mucous layer strengthening effect. The protocol of this explorative pilot study is feasible for a sufficiently powered trial on inflammation amelioration in IBS patients.

Advillin is a tuft cell marker in the mouse alimentary tract.

Tuft cells are a rare population of chemosensory cells at the mucosal surface epithelia of hollow organs. Their name-giving morphological feature is an apical tuft of stiff microvilli. Accordingly, the actin-binding protein, villin, was identified as one of the first tuft cell markers in immunohistochemical analysis. Unfortunately, villin expression is not restricted to tuft cells, but is also prominent e.g. in enterocytes, which limits the use of this gene as a marker and as an experimental tool to genetically target tuft cells. Here, we report that the villin-related protein, advillin, is a specific tuft cell marker in the gastro-intestinal and biliary tract epithelia. In situ hybridization and immunohistochemistry revealed that advillin expression, unlike villin, was restricted to solitary cholinergic tuft cells in the mucosal linings of the small and large intestine, and in the gall bladder. In the glandular stomach, villin and advillin mRNA were present in all epithelial cells, while detectable protein levels were confined to solitary tuft cells. Advillin expression was no longer detectable in the mucosa of the intestinal and biliary tract from Pou2f3 deficient mice that lack tuft cells. Finally, crossing Avil-Cre transgenic mice with a double-fluorescent reporter mouse line resulted in specific targeting of gastro-intestinal and biliary tuft cells. Our analysis introduces advillin as a selective marker and tool in histological and functional analysis of the alimentary tract tuft cell system.