Unveiling an Important New Cell Type in the Lung: Microfold Cells

Mammalian lungs are directly exposed to environmental pathogens and toxins and have evolved complex defense mechanisms, including the mucociliary escalator and secretion of antimicrobial peptides to trap and remove foreign particles and microbes from the airways. Advances in new technologies are now allowing us to identify rare cell types in the respiratory tract that have not been characterized before. In two recent issues of the Journal, studies from two independent groups (1, 2) report on a rare airway epithelial cell type in the lung. It is distinct from other epithelial cells and has gene expression patterns similar to intestinal microfold (M) cells, epithelial cells capable of transcytosis for mucosal immune surveillance (Figure 1).

Figure 1.

Schematic representation of the mouse tracheal epithelium. The tracheal epithelium consists of ciliated, goblet, basal, microfold (M), club, hillock, tuft, and neuroendocrine cells, in addition to ionocytes. Two studies now report rare M cells in the homeostatic mouse trachea (1) and the emergence of M cells in the airways of the mouse lung after influenza infection (2). M cells are specialized epithelial cells that can transport luminal antigens to the underlying immune cells and have been described in the intestine and nasopharynx. Mature M cells are characterized by the expression of glycoprotein-2, which functions as a microbial uptake receptor. B = B lymphocyte; DC = dendritic cell; GP2 = glycoprotein-2; T = T lymphocyte.

M cells are specialized intestinal epithelial cells found in the epithelium covering gut-associated lymphoid tissue, such as the Peyer’s patches in the small intestine (3, 4). M cells take up luminal antigens such as bacteria, viruses, and particles and deliver them to the underlying lymphoid follicles, where they can interact with antigen-presenting cells and B lymphocytes to provide a unique mucosal immune surveillance system. M cells have a distinctive morphology compared with other intestinal epithelial cells in that they have short or absent microvilli on their apical surface and a pocket-like invagination (i.e., M cell pocket) on their basolateral surface that harbors dendritic cells and lymphocytes. M cells constitute 5–10% of the intestinal follicle-associated epithelium in mice and humans (3) and are characterized by several marker genes and proteins. Glycoprotein 2 (GP2) is specifically expressed on mature M cells and functions as a microbial uptake receptor. Marcksl1 (myristoylated alanine rich protein kinase C substrate like-1), Tnfaip2, Anxa5, Ccl9, Spi-B, and Sox8 are also marker genes that are expressed early during M cell differentiation. M cells are derived from intestinal epithelial stem cells, induced by the TNF superfamily cytokine RANKL (receptor activator of NF-κB ligand).

Although M cells are best described in the gut, M cells have been reported in the upper respiratory tract overlying the nasopharynx-associated lymphoid tissue (NALT) (5, 6). Similar to the intestinal M cells, NALT M cells can take up luminal microbes and particles (5–7) and express the mature intestinal M cell marker GP2 as well as other intestinal M cell markers such as Tnfaip2, CCL9, and Spi-B (7). NALT M cells are also induced by RANKL-RANK signaling (7). Unlike the gut or the nasopharynx, the lung has a very low microbial load and was long considered sterile. Nevertheless, a small number of M cells in the mouse trachea do expand upon intraperitoneal administration of RANKL (8). Tracheal M cells are also capable of transcytosis and are spatially associated with pathologically induced bronchus-associated lymphoid tissue (BALT) in mice (8). The cellular origin of these cells and whether M cells exist in the distal airways of the lung remained unknown.

In the recent studies, both groups used single-cell or single-nucleus RNA sequencing and discovered a rare cell type not previously reported in the lung. Surve and colleagues (1) reanalyzed published single-cell RNA sequencing data (9) and revealed the existence of rare M cells in the homeostatic mouse trachea. Tracheal M cells express genes that are characteristic of intestinal M cells, such as Gp2, Tnfaip2, Marcksl1, Anxa5, Sox8, Spib, Ccl9, Ccl20, and Tnfrsf11a (which encodes RANK). Using the mouse tracheal air–liquid interface culture system, Surve and colleagues demonstrated that mature M (GP2⁺) cells are induced from tracheal cells by RANKL, and basal cells and/or secretory cells in the trachea can differentiate into SOX8⁺ cells (early-stage M cell marker) upon RANKL administration in vitro. The uptake of Aspergillus fumigatus conidia by tracheal M cells was dependent on complement receptor 2, which is expressed on tracheal M cells (1).

In comparison, Barr and colleagues (pp. 322–325) report the emergence of M cells in the mouse lung airways after influenza infection (2). These cells share some transcriptomic features with intestinal M cells (Tnfaip2, Sox8, Spib, Ccl9, Ccl20, and Tnfrsf11a) by single-nucleus RNA sequencing analysis and RNAscope imaging. Pulmonary M cells were not observed at baseline without infection. M cell marker gene–positive cells appear in the lung airways starting from 5–11 days post infection (dpi) and persist until 25 dpi, but decrease by 56 dpi. At 25 dpi, Spib ⁺ pulmonary M cells were located in the epithelium overlying BALT. The size of CD19 + BALT was positively correlated with the number of Spib ⁺ pulmonary M cells (2).

Tracheal (1, 8) and pulmonary (2) M cells share some features with their intestinal counterparts, such as the expression of intestinal M cell marker genes (Gp2, Tnfaip2, Sox8, Spib, Ccl9, Ccl20, and Tnfrsf11a) and the dependence on RANKL-RANK signaling for their induction. Tracheal M cells also have the ability to take up luminal materials (1, 8). However, there are some differences between intestinal M cells and tracheal and pulmonary M cells. For example, Gp2, a marker of mature intestinal M cells, is expressed at a higher level in goblet cells than in tracheal and pulmonary M cells in the airway epithelium. Perhaps, tracheal and pulmonary M cells are more similar to GP2-low intestinal M cells, which have a low uptake capacity, than to mature GP2-high intestinal M cells (10). Pulmonary M cells in clusters were observed at 14–25 dpi (2), when most of the influenza virus is cleared (11). The fact that rare pulmonary M cells still exist at 56 dpi suggests that the main function of pulmonary M cells may not be to clear primary pathogens, but to contribute to the diversity of the humoral response to influenza virus by interacting with inducible BALT and B cells (12).

It is exciting to see two independent groups find a novel, previously undescribed cell type in the lung almost simultaneously. One study (2) also provides a comprehensive, high-quality single-nucleus RNA sequencing dataset of airway cells that will be informative for many in the field studying lung responses to viral infection and provides the opportunity to compare postinfluenza pulmonary M cells with homeostatic tracheal M cells from an independent dataset (1). These studies open up new avenues for exploring the role of M cells in lung immunity, infection, and inflammation. Nevertheless, several questions remain unanswered, such as the existence of M cells in the human lung, the ability of pulmonary M cells to transport antigens across the epithelium, and the origin(s) of pulmonary M cells. Therefore, these studies represent an important first step, but not the final word, on the biology of M cells in the lung.