paratuberculosis fibronectin attachment protein facilitates M-cell targeting and invasion through a fibronectin bridge with host integrins. penetrate the mucosal barrier remains unknown. Importantly, about 10% of all cases of active tuberculosis involve isolated infection of cervical lymph nodes (LN) suggesting that pulmonary infection per se may not be required for infection. This form of tuberculosis, also called scrofula commonly manifests in children (Fontanilla et al., 2011). When airborne particles are inhaled, medium and large particles are trapped in the nasopharyngeal and tracheobronchial region, while smaller particles can reach the distal lung (Roy and Milton, 2004). Mtb particles range in size from 0.65 (small) to 7.0 m (medium-large) (Fennelly et al., 2004). Thus, some Mtb-containing particles are likely trapped in the proximal airway while other particles distribute distally. Although the current paradigm is that tuberculosis is initiated by primary infection of alveolar macrophages (AMs), alternative routes of entry such as via epithelial cells or via mucosa-associated lymphatic tissue (MALT) have been proposed (Behr and Waters, 2014; Bermudez and Goodman, (2-Hydroxypropyl)-β-cyclodextrin 1996). Oropharyngeal and airway MALT is prevalent in childhood including nasal-associated lymphatic tissue (NALT), the tonsils and adenoids of Waldeyers ring and bronchus-associated lymphatic tissue (BALT), but tends to regress in adulthood (Debertin et al., 2006). Whether BALT is present in adults is controversial (Randall, 2010); however, BALT-like structures, also known as induced BALT (iBALT) (Foo and Phipps, 2010), can be induced by bacterial and viral infections (Halle et al., 2009). Overlying MALT is a rare, DNM3 specialized cell called a microfold or M-cell whose primary function is to deliver mucosal particles to submucosal antigen presenting cells (Mabbott et al., 2013). M-cells are found primarily in the gastrointestinal tract overlying Peyers patches. M-cells can also be found in the upper airway overlying NALT, adenoids, and BALT. Though significantly less is known about airway than gastrointestinal M-cells (Kanaya and Ohno, 2014), a recent study demonstrated shared (2-Hydroxypropyl)-β-cyclodextrin expression of vital differentiation molecules in mouse NALT M-cells (Mutoh et (2-Hydroxypropyl)-β-cyclodextrin al., 2015). Airway M-cells can mediate infection by bacteria such as (Park et al., 2003) and (Plaut et al., 2012). Infection of cattle with can be achieved by direct inoculation of bovine tonsils (Palmer et al., 2007), and drinking unpasteurized milk contaminated with can cause human tuberculosis that frequently manifests as cervical lymphadenitis (Cosivi et al., 1998). At the cellular level, Mtb (Kumagai, 1922), (BCG) (Fujimura, 1986) and (Secott et al., 2004) can translocate across Peyers patches and it was reported over 15 years ago that in experimentally infected mice Mtb is occasionally found inside cells with the morphologic appearance of M-cells (Teitelbaum et al., 1999). However, a functional analysis of M-cell mediated translocation of Mtb has not been performed. Here we show that airway M-cells can directly mediate primary infection by Mtb and facilitate dissemination beyond the mucosa. Results Genetic depletion of M-cells reduces mycobacterial dissemination to cervical lymph nodes To determine if murine NALT inoculation is sufficient to initiate infection with Mtb, we established an intranasal infection model and found that doses of 106 CFU (2-Hydroxypropyl)-β-cyclodextrin or greater could lead to consistent dissemination to the draining cervical lymph nodes (cLN) 30 days after infection (Supplemental Fig. 1, related to Fig. 1). Using a small volume (10 l) isolated the initial inoculum to the NALT.