Succinate Produced by Intestinal Microbes Promotes Specification of Tuft Cells to Suppress Ileal Inflammation.

BACKGROUND & AIMS: Countries endemic for parasitic infestations have a lower incidence of Crohn's disease (CD) than nonendemic countries, and there have been anecdotal reports of the beneficial effects of helminths in CD patients. Tuft cells in the small intestine sense and direct the immune response against eukaryotic parasites. We investigated the activities of tuft cells in patients with CD and mouse models of intestinal inflammation. METHODS: We used microscopy to quantify tuft cells in intestinal specimens from patients with ileal CD (n = 19), healthy individuals (n = 14), and TNFΔARE/+ mice, which develop Crohn's-like ileitis. We performed single-cell RNA sequencing, mass spectrometry, and microbiome profiling of intestinal tissues from wild-type and Atoh1-knockout mice, which have expansion of tuft cells, to study interactions between microbes and tuft cell populations. We assessed microbe dependence of tuft cell populations using microbiome depletion, organoids, and microbe transplant experiments. We used multiplex imaging and cytokine assays to assess alterations in inflammatory response following expansion of tuft cells with succinate administration in TNFΔARE/+ and anti-CD3E CD mouse models. RESULTS: Inflamed ileal tissues from patients and mice had reduced numbers of tuft cells, compared with healthy individuals or wild-type mice. Expansion of tuft cells was associated with increased expression of genes that regulate the tricarboxylic acid cycle, which resulted from microbe production of the metabolite succinate. Experiments in which we manipulated the intestinal microbiota of mice revealed the existence of an ATOH1-independent population of tuft cells that was sensitive to metabolites produced by microbes. Administration of succinate to mice expanded tuft cells and reduced intestinal inflammation in TNFΔARE/+ mice and anti-CD3E-treated mice, increased GATA3+ cells and type 2 cytokines (IL22, IL25, IL13), and decreased RORGT+ cells and type 17 cytokines (IL23) in a tuft cell-dependent manner. CONCLUSIONS: We found that tuft cell expansion reduced chronic intestinal inflammation in mice. Strategies to expand tuft cells might be developed for treatment of CD.

Zika virus infection in chemosensory cells.

Zika virus (ZIKV) is an emerging virus belonging to the genus Flavivirus. ZIKV infection is a significant health concern, with increasing numbers of reports of microcephaly cases in fetuses and Guillain-Barré syndrome (GBS) in adults. Interestingly, chemosensory disturbances are also reported as one of the manifestations of GBS. ZIKV infects several human tissues and cell types in vitro and in vivo. However, there is no study demonstrating ZIKV infection and replication in chemosensory cells, including olfactory and taste cells. Taste papilla and olfactory cells are chemosensory receptor cells with unique histological, molecular, and physiological characteristics. Here we examined ZIKV infection (PRVABC59) in cultured human olfactory epithelial cells (hOECs) and fungiform taste papilla (HBO) cells in vitro, as well as in vivo mouse taste and olfactory epithelial and olfactory bulb tissues. Interestingly, while HBO cells showed resistance to ZIKV replication, hOECs were highly susceptible for ZIKV infection and replication. Further, we demonstrated the presence of ZIKV particles and expression of viral proteins in olfactory epithelium, as well as in olfactory bulb, but not in taste papillae, of immunocompromised mice (ifnar/-) infected with the PRVABC59 strain of ZIKV. These observations suggest that chemosensory cells in the olfactory neuroepithelium and olfactory bulb may be important tissues for ZIKV replication and dissemination.

Gingival solitary chemosensory cells are immune sentinels for periodontitis.

Solitary chemosensory cells (SCCs) are epithelial sentinels that utilize bitter Tas2r receptors and coupled taste transduction elements to detect pathogenic bacterial metabolites, triggering host defenses to control the infection. Here we report that SCCs are present in mouse gingival junctional epithelium, where they express several Tas2rs and the taste signaling components α-gustducin (Gnat3), TrpM5, and Plcß2. Gnat3-/- mice have altered commensal oral microbiota and accelerated naturally occurring alveolar bone loss. In ligature-induced periodontitis, knockout of taste signaling molecules or genetic absence of gingival SCCs (gSCCs) increases the bacterial load, reduces bacterial diversity, and renders the microbiota more pathogenic, leading to greater alveolar bone loss. Topical treatment with bitter denatonium to activate gSCCs upregulates the expression of antimicrobial peptides and ameliorates ligature-induced periodontitis in wild-type but not in Gnat3-/- mice. We conclude that gSCCs may provide a promising target for treating periodontitis by harnessing innate immunity to regulate the oral microbiome.

Fungal extracts stimulate solitary chemosensory cell expansion in noninvasive fungal rhinosinusitis.

BACKGROUND: Solitary chemosensory cells (SCCs) are rare epithelial cells enriched in nasal polyps and are the primary source of interleukin-25 (IL-25), an innate cytokine eliciting T-helper 2 (Th2) immune response. Although it is proposed that SCCs are stimulated by antigens released by upper airway pathogens, the exogenous triggers of human SCCs remain elusive. We studied patients with noninvasive fungal rhinosinusitis to determine whether extracts of Aspergillus fumigatus and Alternaria alternata stimulate SCC proliferation as an early event in type 2 inflammation. METHODS: Multicolor flow cytometry, immunofluorescence, and enzyme-linked immunoassay were used to interrogate mucosa from patients with mycetomas and allergic fungal rhinosinusitis (AFRS) for SCCs and IL-25. Primary sinonasal epithelial cells from AFRS patients and noninflamed inferior turbinates were stimulated with fungal extracts for 72 hours, and SCC population frequency as well as mitotic activity were quantified using flow cytometry. RESULTS: SCCs producing IL-25 are enriched in inflamed mucosa compared with intrapatient noninflamed control tissue (38.6% vs 6.5%, p = 0.029). In cultured sinonasal epithelial cells from AFRS nasal polyps, Aspergillus fumigatus and Alternaria alternata stimulated higher SCC frequency compared with controls (27.4% vs 10.6%, p = 0.002; 18.1% vs 10.6%, p = 0.046), which led to increased IL-25 secretion in culture media (75.5 vs 3.3 pg/mL, p < 0.001; 32.3 vs 3.3 pg/mL, p = 0.007). Ki-67 expression was higher in SCCs grown in fungal stimulation conditions compared with controls. CONCLUSION: Although fungal antigens are known to potentiate immune response through innate cytokines, including IL-25, the early expansion of SCCs in the presence of fungus has not been described. This early event in the pathogenesis of noninvasive fungal rhinosinusitis may represent a target for intervention.

Bacterial d-amino acids suppress sinonasal innate immunity through sweet taste receptors in solitary chemosensory cells.

In the upper respiratory epithelium, bitter and sweet taste receptors present in solitary chemosensory cells influence antimicrobial innate immune defense responses. Whereas activation of bitter taste receptors (T2Rs) stimulates surrounding epithelial cells to release antimicrobial peptides, activation of the sweet taste receptor (T1R) in the same cells inhibits this response. This mechanism is thought to control the magnitude of antimicrobial peptide release based on the sugar content of airway surface liquid. We hypothesized that d-amino acids, which are produced by various bacteria and activate T1R in taste receptor cells in the mouth, may also activate T1R in the airway. We showed that both the T1R2 and T1R3 subunits of the sweet taste receptor (T1R2/3) were present in the same chemosensory cells of primary human sinonasal epithelial cultures. Respiratory isolates of Staphylococcus species, but not Pseudomonas aeruginosa, produced at least two d-amino acids that activate the sweet taste receptor. In addition to inhibiting P. aeruginosa biofilm formation, d-amino acids derived from Staphylococcus inhibited T2R-mediated signaling and defensin secretion in sinonasal cells by activating T1R2/3. d-Amino acid-mediated activation of T1R2/3 also enhanced epithelial cell death during challenge with Staphylococcus aureus in the presence of the bitter receptor-activating compound denatonium benzoate. These data establish a potential mechanism for interkingdom signaling in the airway mediated by bacterial d-amino acids and the mammalian sweet taste receptor in airway chemosensory cells.

Tuft cells, taste-chemosensory cells, orchestrate parasite type 2 immunity in the gut.

The intestinal epithelium forms an essential barrier between a host and its microbiota. Protozoa and helminths are members of the gut microbiota of mammals, including humans, yet the many ways that gut epithelial cells orchestrate responses to these eukaryotes remain unclear. Here we show that tuft cells, which are taste-chemosensory epithelial cells, accumulate during parasite colonization and infection. Disruption of chemosensory signaling through the loss of TRMP5 abrogates the expansion of tuft cells, goblet cells, eosinophils, and type 2 innate lymphoid cells during parasite colonization. Tuft cells are the primary source of the parasite-induced cytokine interleukin-25, which indirectly induces tuft cell expansion by promoting interleukin-13 production by innate lymphoid cells. Our results identify intestinal tuft cells as critical sentinels in the gut epithelium that promote type 2 immunity in response to intestinal parasites.

Pulmonary chemoreflex responses are potentiated by tumor necrosis factor-alpha in mice.

Inhalation of tumor necrosis factor-alpha (TNF-α), a proinflammatory cytokine, induces airway hyperresponsiveness, and the underlying mechanism is not fully understood. Hypersensitivity of vagal bronchopulmonary C-fiber afferents is known to contribute to the airway hyperresponsiveness during an airway inflammatory reaction. Because activation of these afferents can elicit pulmonary chemoreflexes, this study was designed to determine if a pretreatment with TNF-α induced airway inflammation and enhanced the pulmonary chemoreflex sensitivity in anesthetized mice; and if so, whether the effect was mediated through activation of either or both of the TNF receptors, p55 and p75. Our results showed that TNF-α instilled into the lung caused an increased sensitivity of pulmonary chemoreflex responses to various chemical stimulants of the vagal bronchopulmonary C-fiber afferents. The increased sensitivity was found 24 h later, persisted at 48 h, and then gradually declined after several days. The TNF-α-induced airway hypersensitivity was accompanied by airway inflammation as shown by a striking elevation of the levels of eosinophils and neutrophils, several potent bronchoactive inflammatory mediators, and proinflammatory cytokines in the bronchoalveolar lavage fluid. Furthermore, the increase in pulmonary chemoreflex response caused by TNF-α was partially abrogated in both p55-null and p75-null mice, but completely abolished in p55/p75-null mice. In conclusion, TNF-α pretreatment induced airway inflammation and a sustained elevation of pulmonary chemoreflex sensitivity, which was mediated through an activation of both types of TNF receptors.

Inflammatory and immunomodulatory mechanisms in the carotid body.

Evidence is available about the role of inflammatory/immunological factors in the physiology and plasticity of the carotid body, with potential clinical implications in obstructive sleep apnea syndrome and sudden infant death syndrome. In humans, lymphomonocytic aggregations (chronic carotid glomitis) have been reported in aging and opiate addiction. Glomus cells produce prostaglandin E2 and the cytokines interleukin 1ß, interleukin 6 and TNF-α, with corresponding receptors. These factors modulate glomus cell excitability, catecholamine release and/or chemoreceptor discharge. The above cytokines are up-regulated in chronic sustained or intermittent hypoxia, and prevention of these changes, with ibuprofen or dexamethasone, may modulate hypoxia-induced changes in carotid body chemosensitivity. The main transcription factors considered to be involved are NF-kB and HIFs. Circulating immunogens (lipopolysaccharide) and cytokines may also affect peripheral arterial chemoreception, with the carotid body exerting an immunosensing function.

Immunosensory signalling by carotid body chemoreceptors.

Injections of lipopolysaccharide (LPS) have been used to produce the signs of sepsis and study their underlying mechanisms. Intravenous (IV) injections of LPS in anesthetized cats induce tachypnea, tachycardia and hypotension, but ventilatory changes are suppressed after sectioning carotid and aortic nerves. Otherwise, LPS increases the basal frequency of carotid chemosensory discharges, but reduces ventilatory and chemosensory responses to hypoxia and nicotine injections. Increases in cytokines (IL-1ß, IL-6 and TNF-α) are observed in plasma and tissues after injecting LPS. In carotid bodies perfused in vitro, TNF-α reduces chemosensory discharges induced by hypoxia. The rat carotid body and its sensory ganglion constitutively express LPS canonical receptor, TLR4, as well as TNF-α and its receptors (TNF-R1 and TNF-R2). Increases of TNF-α and TNF-R2 expression occur after LPS administration. The activation of peripheral and central autonomic pathways induced by LPS or IL's is partly dependent on intact vagus nerves. Thus, the carotid and vagus nerves provide routes between the immune system and CNS structures involved in systemic inflammatory responses.

Nasonasal reflexes, the nasal cycle, and sneeze.

The nasal mucosa is a complex tissue that interacts with its environment and effects local and systemic changes. Receptors in the nose receive signals from stimuli, and respond locally through afferent, nociceptive, type C neurons to elicit nasonasal reflex responses mediated via cholinergic neurons. This efferent limb leads to responses in the nose (eg, rhinorrhea, glandular hyperplasia, hypersecretion with mucosal swelling). Anticholinergic agents appear useful against this limb for symptomatic relief of a "runny nose." Chronic exposure to allergens can lead to hyperresponsiveness of the nasal mucosa. As a result, receptors upregulate specific ion channels to increase the sensitivity and potency of their reflex response. Nasal stimuli also affect distant parts of the body. Nerves in the sinus mucosa cause vasodilation; the lacrimal glands can be stimulated by nasal afferent triggers. Even the cardiopulmonary system can be affected via the trigeminal chemosensory system, where sensed irritants can lead to changes in tidal volume, respiratory rate, and blink frequency. The sneeze is an airway defense mechanism that removes irritants from the nasal epithelial surface. It is generally benign, but can lead to problems in certain circumstances. The afferent pathway involves histamine-mediated depolarization of H1 receptor-bearing type C trigeminal neurons and a complex coordination of reactions to effect a response.

Female choice and the MHC.

In animals, it is the female that typically selects a mating partner. This decision can occur before, during and after copulation. Here, recent evidence for the involvement of genes within the MHC in female choice is reviewed and the roles of MHC I and II antigens, various types of chemoreceptors, as well as MHC-encoded transcription factors, in securing an optimal genetic constitution of the offspring are discussed. Some particularly interesting and as yet unanswered questions are raised and some experiments that could provide deeper insight into the molecular mechanisms underlying female choice are suggested.

Triple immunofluorescence staining with antibodies raised in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors.

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method when primary antibodies must be used that originate in the same species. We have developed a protocol for the immunodetection of three antigens in a single tissue preparation, using unconjugated primary antibodies raised in the same species. Immunocytochemical detection of neuronal nitric oxide synthase, calcitonin gene-related peptide, and calbindin D28k in the lung of rats demonstrated that part of the pulmonary neuroepithelial bodies are selectively contacted by at least three different nerve fiber populations. The first antigen was detected using tyramide signal amplification, a very sensitive method allowing a dilution of the first primary antibody far beyond the detection limit of fluorescently labeled secondary antibodies. The second antigen was visualized by a fluorophore-conjugated secondary monovalent Fab antibody that at the same time blocks the access of the third secondary antibody to the second primary antibody. Moreover, the monovalence of the Fab fragment prevents the third primary antibody from binding with the second-step secondary antibody. The triple staining technique described here is generally applicable, uses commercially available products only, and allows the detection of three antigens in the same preparation with primary antibodies that are raised in the same species.

Gustatory organs of Drosophila melanogaster: fine structure and expression of the putative odorant-binding protein PBPRP2.

In Drosophila, as in most insects, gustation is mediated by sensory hairs located on the external and internal parts of the proboscis and on the legs and wings. We describe in detail the organization and ultrastructure of the gustatory sensilla on the labellum and legs and the distribution of PBPRP2, a putative odorant-binding protein, in the gustatory organs of Drosophila. The labellum carries two kinds of sensilla: taste bristles and taste pegs. The former have the typical morphology of gustatory sensilla and can be further subdivided into three morphological subtypes, each with a stereotyped distribution and innervation. Taste pegs have a unique morphology and are innervated by two receptor cells: one mechanoreceptor and the other a putative chemoreceptor cell. PBPRP2 is abundantly expressed in all adult gustatory organs on labellum, legs, and wings and in the internal taste organs on the proboscis. In contrast to olfactory organs, where PBPRP2 is expressed in the epidermis, this protein is absent from the epidermis of labial palps and legs. In the taste bristles of the labellum and legs, PBPRP2 is localized in the crescent-shaped lumen of the sensilla, and not in the lumen where the dendrites of the gustatory neurons are found, making a function in stimulus transport unlikely in these sensilla. In contrast, PBPRP2 in peg sensilla is expressed in the inner sensillum-lymph cavity and is in contact with the dendrites. Thus, PBPRP2 could be involved as a carrier for hydrophobic ligands, e.g., bitter tastants, in these sensilla.

Vagal immune-to-brain communication: a visceral chemosensory pathway.

The immune system operates as a diffuse sensory system, detecting the presence of specific chemical constituents associated with dangerous micro-organisms, and then signalling the brain. In this way, immunosensation constitutes a chemosensory system. Several submodalities of this sensory system function as pathways conveying immune-related information, and can be classified as either primarily brain barrier associated or neural. The vagus nerve provides the major neural pathway identified to date. The initial chemosensory transduction events occur in immune cells, which respond to specific chemical components expressed by dangerous micro-organisms. These immune chemosensory cells release mediators, such as cytokines, to activate neural elements, including primary afferent neurons of the vagal sensory ganglia. Primary afferent activation initiates local reflexes (e.g. cardiovascular and gastrointestinal) that support host defense. In addition, at least three parallel pathways of ascending immune-related information activate specific components of the illness response. In this way, immunosensory systems represent highly organized and coherent pathways for activating host defense against infection.

Possible active site of the sweet-tasting protein thaumatin.

Epitopes on thaumatin and monellin were studied using the PEPSCAN-technology. The antibodies used were raised against thaumatin. Only antibodies that, in an ELISA, both recognized thaumatin and monellin were used in the PEPSCAN-analyses. On thaumatin two major overlapping epitopes were identified. On monellin no epitopes could be identified. The identified epitope region on thaumatin shares structural features with various peptide and protein sweeteners. It contains an aspartame-like site which is formed by Asp21 and Phe80, tips of the two extruding loops KGDAALDAGGR19-29 and CKRFGRPP77-84, which are spatially positioned next to each other. Furthermore, sub-sequences of the KGDAALDAGGR19-29 loop are similar to peptide-sweeteners such as L-Asp-D-Ala-L-Ala-methyl ester and L-Asp-D-Ala-Gly-methyl ester. Since the aspartame-like Asp21-Phe80 site and the peptide-sweetener-like sequences are also not present in non-sweet thaumatin-like proteins it is postulated that the KGDAALDAGGR19-29- and CKRFGRPP77-84 loop contain important sweet-taste determinants. This region has previously not been implicated as a sweet-taste determinant of thaumatin.

Calretinin, calbindin-D28k and parvalbumin-like immunoreactivity in mouse chemoreceptor neurons.

Calretinin immunoreactivity was demonstrated in adult mouse olfactory receptor neurons and in the vomeronasal and septal chemoreceptor neurons, whereas parvalbumin expression was restricted to the vomeronasal receptor neurons. Calbindin-D28k-like immunoreactivity was primarily localized in the vomeronasal and septal chemoreceptor neurons although an occasional neuronal staining with calbindin-D28k was also found in restricted areas of the main olfactory epithelium.

Generation of monoclonal antibodies detecting specific epitopes in locust antennae.

1. Following a tissue-specific screening paradigm, monoclonal antibodies have been generated that interact with distinct subpopulations of cells in locust antennae. 2. Antigens were identified as high molecular weight components. 3. Immunoreactivity was not detectable during embryonic development, but rapidly appeared within a few hours of hatching. 4. The time course of antigen expression in antennal cells could be followed in situ as well as in vitro. 5. Expression of monoclonal antibody B14/6D2-like immunoreactivity was prevented by blocking protein synthesis with cycloheximide.

Immunohistochemical study of the fourth cell type in the olfactory epithelium in guinea pigs and in a patient.

The mammalian olfactory epithelium consists of supporting cells, olfactory receptor cells, basal cells and a fourth cell type, which has recently been discovered. In this study we examine this fourth cell type using immunohistochemical techniques. Anti-Purkinje-specific spot-35 protein (S-35), anti-S-100 protein (S-100), anti-neuron-specific enolase (NSE), antichromogranin A and antisynaptophysin antisera were used for the immunostaining. The fourth cell type immunoreacted only to anti-S-35 antiserum and did not react to other antisera in guinea pigs. On the other hand, in the human S-100 immunoreactivity was seen in the fourth cell type as well as weak reactivity to S-35. NSE immunoreactivity was found only in the olfactory receptor cells of guinea pigs and the human. From these results it is assumed that this fourth cell type is a second chemoreceptor different from the olfactory receptor cells because S-35 and S-100 are Ca(++)-binding proteins.