A nasal cell atlas reveals heterogeneity of tuft cells and their role in directing olfactory stem cell proliferation.

The olfactory neuroepithelium serves as a sensory organ for odors and forms part of the nasal mucosal barrier. Olfactory sensory neurons are surrounded and supported by epithelial cells. Among them, microvillous cells (MVCs) are strategically positioned at the apical surface, but their specific functions are enigmatic, and their relationship to the other specialized epithelial cells is unclear. Here, we establish that the family of MVCs comprises tuft cells and ionocytes in both mice and humans. Integrating analysis of the respiratory and olfactory epithelia, we define the distinct receptor expression of TRPM5+ tuft-MVCs compared with Gɑ-gustducinhigh respiratory tuft cells and characterize a previously undescribed population of glandular DCLK1+ tuft cells. To establish how allergen sensing by tuft-MVCs might direct olfactory mucosal responses, we used an integrated single-cell transcriptional and protein analysis. Inhalation of Alternaria induced mucosal epithelial effector molecules including Chil4 and a distinct pathway leading to proliferation of the quiescent olfactory horizontal basal stem cell (HBC) pool, both triggered in the absence of olfactory apoptosis. Alternaria- and ATP-elicited HBC proliferation was dependent on TRPM5+ tuft-MVCs, identifying these specialized epithelial cells as regulators of olfactory stem cell responses. Together, our data provide high-resolution characterization of nasal tuft cell heterogeneity and identify a function of TRPM5+ tuft-MVCs in directing the olfactory mucosal response to allergens.

Isolation, Ex Vivo Culture, and Stimulation of Tracheal and Nasal Chemosensory Cells.

Brush cells are chemosensory epithelial cells present at most mucosal surfaces.Brush cells are a dominant source of cysteinyl leukotrienes and IL-25 in the airway epithelium and are equipped with the machinery to generate prostaglandins and acetylcholine. Activation of innate type 2 lymphoid cells and dendritic cells triggered by brush cell-derived mediators skew the immune response in the airway to type 2 inflammation that underlies atopic disease such as asthma. This chapter describes an effective method of brush cell isolation from the mouse trachea for transcriptional analysis and from the nasal cavity for transcriptional analysis and ex vivo stimulation.The nasal or tracheal mucosa is first incubated in a dispase solution for easy mechanical separation of the epithelial layer from the underlying submucosa. The detached epithelium is then digested with a papain solution. This method provides high yields of viable brush cells in a single-cell suspension, which can be used for flow cytometric analysis, single-cell sorting, cell culture, and functional assays.In the nose, where brush cells are more abundant, we present two methods of isolation of brush cells: (1) using fluorescent reporter mice that mark brush cells or (2) using a combination of high expression of EpCAM and low expression of CD45 to obtain a population of cells that is enriched for nasal chemosensory brush cells.

Tuft cell-produced cysteinyl leukotrienes and IL-25 synergistically initiate lung type 2 inflammation.

Aeroallergen sensing by airway epithelial cells triggers pathogenic immune responses leading to type 2 inflammation, the hallmark of chronic airway diseases such as asthma. Tuft cells are rare epithelial cells and the dominant source of interleukin-25 (IL-25), an epithelial cytokine, and cysteinyl leukotrienes (CysLTs), lipid mediators of vascular permeability and chemotaxis. How these two mediators derived from the same cell might cooperatively promote type 2 inflammation in the airways has not been clarified. Here, we showed that inhalation of the parent leukotriene C4 (LTC4) in combination with a subthreshold dose of IL-25 led to activation of two innate immune cells: inflammatory type 2 innate lymphoid cell (ILC2) for proliferation and cytokine production, and dendritic cells (DCs). This cooperative effect led to a much greater recruitment of eosinophils and CD4+ T cell expansion indicative of synergy. Whereas lung eosinophilia was dominantly mediated through the classical CysLT receptor CysLT1R, type 2 cytokines and activation of innate immune cells required signaling through CysLT1R and partially CysLT2R. Tuft cell­specific deletion of Ltc4s, the terminal enzyme required for CysLT production, reduced lung inflammation and the systemic immune response after inhalation of the mold aeroallergen Alternaria; this effect was further enhanced by concomitant blockade of IL-25. Our findings identified a potent synergy of CysLTs and IL-25 downstream of aeroallergen-trigged activation of airway tuft cells leading to a highly polarized type 2 immune response and further implicate airway tuft cells as powerful modulators of type 2 immunity in the lungs.

Isolation of Nasal Brush Cells for Single-cell Preparations.

Solitary chemosensory epithelial cells are scattered in most mucosal surfaces. They are referred to as tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. They are the primary source of IL-25 in the epithelium and are also engaged in acetylcholine generation. We recently demonstrated that nasal solitary chemosensory (brush) cells can generate robust levels of cysteinyl leukotrienes in response to stimulation with calcium ionophore, aeroallergens, and danger-associated molecules, such as ATP and UTP, and this mechanism depends on brush cell expression of the purinergic receptor P2Y2. This protocol describes an effective method of nasal brush cell isolation in the mouse. The method is based on physical separation of the mucosal layer of the nasal cavity and pre-incubation with dispase, followed by digestion with papain solution. The single cell suspension obtained this way contains a high yield of brush cells for fluorescence-activated cell sorting (FACS), RNA-sequencing, and ex vivo assays. Graphic abstract: Workflow of nasal digestion for brush cell isolation.

The CysLT2R receptor mediates leukotriene C4-driven acute and chronic itch.

Acute and chronic itch are burdensome manifestations of skin pathologies including allergic skin diseases and atopic dermatitis, but the underlying molecular mechanisms are not well understood. Cysteinyl leukotrienes (CysLTs), comprising LTC4, LTD4, and LTE4, are produced by immune cells during type 2 inflammation. Here, we uncover a role for LTC4 and its signaling through the CysLT receptor 2 (CysLT2R) in itch. Cysltr2 transcript is highly expressed in dorsal root ganglia (DRG) neurons linked to itch in mice. We also detected CYSLTR2 in a broad population of human DRG neurons. Injection of leukotriene C4 (LTC4) or its nonhydrolyzable form NMLTC4, but neither LTD4 nor LTE4, induced dose-dependent itch but not pain behaviors in mice. LTC4-mediated itch differed in bout duration and kinetics from pruritogens histamine, compound 48/80, and chloroquine. NMLTC4-induced itch was abrogated in mice deficient for Cysltr2 or when deficiency was restricted to radioresistant cells. Itch was unaffected in mice deficient for Cysltr1, Trpv1, or mast cells (WSh mice). CysLT2R played a role in itch in the MC903 mouse model of chronic itch and dermatitis, but not in models of dry skin or compound 48/80- or Alternaria-induced itch. In MC903-treated mice, CysLT levels increased in skin over time, and Cysltr2-/- mice showed decreased itch in the chronic phase of inflammation. Collectively, our study reveals that LTC4 acts through CysLT2R as its physiological receptor to induce itch, and CysLT2R contributes to itch in a model of dermatitis. Therefore, targeting CysLT signaling may be a promising approach to treat inflammatory itch.

Airway brush cells generate cysteinyl leukotrienes through the ATP sensor P2Y2.

Chemosensory epithelial cells (EpCs) are specialized cells that promote innate type 2 immunity and protective neurally mediated reflexes in the airway. Their effector programs and modes of activation are not fully understood. Here, we define the transcriptional signature of two choline acetyltransferase-expressing nasal EpC populations. They are found in the respiratory and olfactory mucosa and express key chemosensory cell genes including the transcription factor Pou2f3, the cation channel Trpm5, and the cytokine Il25 Moreover, these cells share a core transcriptional signature with chemosensory cells from intestine, trachea and thymus, and cluster with tracheal brush cells (BrCs) independently from other respiratory EpCs, indicating that they are part of the brush/tuft cell family. Both nasal BrC subsets express high levels of transcripts encoding cysteinyl leukotriene (CysLT) biosynthetic enzymes. In response to ionophore, unfractionated nasal BrCs generate CysLTs at levels exceeding that of the adjacent hematopoietic cells isolated from naïve mucosa. Among activating receptors, BrCs express the purinergic receptor P2Y2. Accordingly, the epithelial stress signal ATP and aeroallergens that elicit ATP release trigger BrC CysLT generation, which is mediated by the P2Y2 receptor. ATP- and aeroallergen-elicited CysLT generation in the nasal lavage is reduced in mice lacking Pou2f3, a requisite transcription factor for BrC development. Last, aeroallergen-induced airway eosinophilia is reduced in BrC-deficient mice. These results identify a previously undescribed BrC sensor and effector pathway leading to generation of lipid mediators in response to luminal signals. Further, they suggest that BrC sensing of local damage may provide an important sentinel immune function.

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas.

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

The cysteinyl leukotriene 3 receptor regulates expansion of IL-25-producing airway brush cells leading to type 2 inflammation.

Respiratory epithelial cells (EpCs) orchestrate airway mucosal inflammation in response to diverse environmental stimuli, but how distinct EpC programs are regulated remains poorly understood. Here, we report that inhalation of aeroallergens leads to expansion of airway brush cells (BrCs), specialized chemosensory EpCs and the dominant epithelial source of interleukin-25 (IL-25). BrC expansion was attenuated in mice lacking either LTC4 synthase, the biosynthetic enzyme required for cysteinyl leukotriene (CysLT) generation, or the EpC receptor for leukotriene E4 (LTE4), CysLT3R. LTE4 inhalation was sufficient to elicit CysLT3R-dependent BrC expansion in the murine airway through an IL-25-dependent but STAT6-independent signaling pathway. Last, blockade of IL-25 attenuated both aeroallergen and LTE4-elicited CysLT3R-dependent type 2 lung inflammation. These results demonstrate that CysLT3R senses the endogenously generated lipid ligand LTE4 and regulates airway BrC number and function.