Background

Brush cells are chemosensory epithelial cells best known for their expression of bitter taste receptors (Finger et al., 2003; Krasteva and Kummer, 2012). Brush cells are also be referred to as tuft cells and have been found in the airways (Krasteva and Kummer, 2012), gastrointestinal tract (Howitt et al., 2016), and urinary epithelium (Deckmann et al., 2014). Morphologically, brush cells are characterized by apical microvilli that form a tuft-like projection on the cell surface, extending to the mucosal lumen (Rhodin and Dalhamn, 1956; Reid et al., 2005). Protective functions of brush cells have been most closely linked to their generation of acetylcholine leading to activation of peptidergic sensory nerve fibers (Krasteva et al., 2011). We have previously reported a role for brush cell-derived IL-25 in eliciting type 2 inflammation and epithelial cell remodeling in the lung (Bankova et al., 2018). We recently reported robust generation of pro-inflammatory lipid mediators termed cysteinyl leukotrienes by nasal brush cells in response to aeroallergens and ATP through the purinergic receptor P2Y2 (Ualiyeva et al., 2020), suggesting potent functions for these cells in directing immune responses in the airways.

In the nose, the mucosal lining is composed of olfactory and respiratory epithelia (Adams, 1972). In mice, the olfactory epithelium lines up to 50% of the total nasal mucosa, overlying the middle and superior turbinates and the caudal/posterior area of the nasal septum, while the respiratory epithelium covers around 45% of the nasal cavity in more rostral/anterior parts (Gross et al., 1982; Chamanza and Wright, 2015). Solitary chemosensory cells in the respiratory mucosa of the nose respond to irritants and bacteria and are established as the nasal equivalent of tracheal brush cells in mice (Finger et al., 2003; Sbarbati and Osculati, 2003) and humans (Lee et al., 2014; Kohanski et al., 2018). Microvillous cells are related cells found in the apical olfactory epithelium, above the layer of olfactory sensory neurons, and supporting sustentacular cells (Hansen and Finger, 2008). Both microvillous cells and solitary chemosensory cells express Chat, choline acetyltransferase, an enzyme necessary for the synthesis of the neurotransmitter acetylcholine (Krasteva et al., 2011), Trpm5, a transient receptor potential gene ubiquitous in chemosensory cells (Pérez et al., 2002; Kaske et al., 2007; Zhang et al., 2007; Ogura et al., 2011; Genovese and Tizzano, 2018), and depend on Pou2f3, a transcription factor required for differentiation of all chemosensory cells in the nose, trachea, and intestine (Ohmoto et al., 2013; Yamaguchi et al., 2014; von Moltke et al., 2016).

Our recent study using fluorescence-activated cell sorting (FACS) based on the fluorescent reporter expression of Chat (ChAT-eGFP) and size and granularity characteristics [forward scatter (FSC) and side scatter (SSC)] allowed us to distinguish the olfactory-enriched microvillar cells from the respiratory-enriched solitary chemosensory cells. We confirmed by bulk RNA-sequencing (RNA-seq) that both populations belong to the larger family of chemosensory tuft/brush cells and are mostly distinguished by their varied expression of taste receptors. This protocol describes our method for isolation of a mixed population of nasal chemosensory brush cells providing high yields of ~20,000/mouse, which can be further characterized by FACS and RNA-seq for investigation of the similarities and differences between these cell types in the nasal cavity.

Several groups have reported successful isolation of solitary chemosensory cells from the respiratory nasal mucosa and microvillar cells from the olfactory mucosa. Gulbransen et al. (2008) used a Tyrode-based solution supplemented with 20 U/ml of papain to isolate solitary chemosensory cells from the respiratory mucosa of TRPM5-GFP fluorescent reporter mice. Several hundred viable cells were used for calcium flux studies in response to bitter tasting agonists. Lin et al. (2008) isolated solitary chemosensory cells from the anterior respiratory epithelium with a Ringer-based solution with 10-30 U/ml of papain to measure changes in intracellular calcium levels in single cells in response to odorous irritants. Ogura et al. (2011) isolated microvillar cells from the main olfactory epithelium of ChAT-eGFP and TRPM5-GFP mice with 4 U/ml of papain for 3-5 min at room temperature and showed increased calcium flux from freshly isolated cells in response to ATP, soil bacterium lysate, and denatonium benzoate. Although no information was provided for the total yields of recovered solitary chemosensory or microvillous cells with the described protocols, each used <100 cells for live imaging for calcium flux. We tested a comparable concentration of papain (26 U/ml) to isolate brush cells from the trachea and found that pre-incubation of the trachea with high dose dispase (16 U/ml) results in a 20-fold increase of isolated brush cell yields compared to digestion with papain alone (Ualiyeva et al., 2019).

Here, we describe a step-by-step protocol for isolation of brush cells from the murine snout of ChAT-eGFP fluorescent transgenic mice, with enhanced green fluorescent protein expression dictated by Chat. Choline acetyltransferase is highly expressed in both subsets of nasal chemosensory brush cells, in tracheal, intestinal, and urethral brush/tuft cells and specifically enriched in these chemosensory epithelial cells compared to other epithelial cells. This allows for identification of the fluorescent cholinergic brush cells by FACS. The single-cell solution preparation procedure is based on initial incubation of the snout with dispase solution, which allows for easy mechanical separation of the mucosa from the underlying bones and cartilages. The sample is subsequently incubated with 26 U/ml papain solution for 40 min at 37°C with agitation, which delivers fine dissociation of tight and adherens junctions between epithelial cells. This method grants access to a significant number of viable cells in a single-cell suspension for FACS sorting, RNA-seq, and functional assays.