In contrast to untreated granules, protease inhibitor cocktail-treated MC granules remained strictly within LAMP1+compartments with limited seepage into the cytosol (Figure 5C, right). response to microbial contamination at mucosal surfaces is exfoliation of pathogen-laden epithelial cells. When epithelial cells become overburdened with pathogenic bacteria, they initiate self-destruction processes resulting in reduced microbial burden (Jones et al., 1997). However , this process cannot occur indiscriminately as JTV-519 free base these epithelial cells also serve an important barrier function protecting the underlying cells from potentially toxic substances in the lumen of the mucosal JTV-519 free base tract. Therefore , it is sensible to expect a regulatory mechanism to coordinate this critical activity. Among the best examples of epithelial cell shedding following microbial infection happens in the urinary tract. Urinary tract infections (UTIs) are the second most common bacterial infections in humans (Foxman, 2010) as well as treatment and management especially when recurrent or chronic is increasingly a challenge. UTIs are primarily caused by uropathogenicE. coli(UPEC) that gain access to the bladder and rapidly invade superficial bladder epithelial cells (BECs) to avoid being flushed out when urine is voided. The bladder epithelium is a stratified epithelial structure with a luminal layer comprised of fully differentiated and usually binucleated cells (Hicks, 1975). Underneath the superficial cells lay intermediate cells, which have a limited capacity to contact form tight junctions, followed by basal cells, which are largely responsible for the quick cell proliferation that occurs following the exfoliation of overlying cells (Mysorekar et al., 2009). When superficial BECs become heavily infected by uropathogens, they spontaneously lose their viability and sheden masseinto the urine, a reaction reported to be bacteria-triggered apoptosis (Mulvey et al., 1998). Pyroptosis is a distinct and a more recently found out form of cell death in response to contamination JTV-519 free base which up to now has mainly been observed in macrophages and other hematopoietic cells following bacterial infection (Bergsbaken et al., 2009). A large cytosolic protein complex structure called the inflammasome mediates pyroptosis by evoking the cleavage and activation of caspase-1, a mediator of cytolysis. The inflammasome mediates several other important innate immune responses to microbial challenge including the maturation and secretion of proinflammatory cytokines interleukin-1 (IL-1) and IL-18 (Bergsbaken et al., 2009). The nature of the cellular immune response mediated by the inflammasome is often dictated by the type of complexes it forms in the subcellular milieu following activation (Broz et al., 2010; Chen et al., 2014). Here, we investigated the possibility that UPEC induced exfoliation of BECs involved inflammasome reliant cytolysis because of our observation that exfoliated BECs from infected mice had undergone lytic cell death. Although inflammasome was implicated, its role in BEC lysis and exfoliation was indirect and lytic cell death required the collaboration of recently recruited MCs. These cells are the source of a critical cytolytic signal that determine which and when infected epithelial cells are shed following infection and this requirement appears be a web host mechanism to prevent inappropriate and potentially harmful exfoliation of epithelial cells. == Results == == Dynamics of infection-induced shedding and loss in BEC viability == To explore BEC exfoliation, we characterized its dynamics in a murine UTI model. Following catheter mediated instillation of UPEC strain CI5, a frequently used and recently sequenced pyelonephritis isolate (Mehershahi et al., 2015; Song et al., 2007), into the bladders of wild-type (WT) C57BL/6 mice, the bulk of BEC exfoliated between 6 and 12 h post-inoculation. Microscopic examination of mouse bladder mix sections revealed that superficial BECs were mainly intact at 6 h, but their widespread loss was visually evident by 12 h post-infection (Figure 1A). This obtaining was supported by quantitation of residual superficial BECs on whole mounts of bladder tissue examined at 6 and 12 h post-infection or after urethral administration of saline control. By 12 h post-infection, up to 75% of superficial BECs had been lost compared to RHOC saline regulates (Figure 1B). Because lack of superficial BECs has been linked to a reduction in bacterial burden, we quantitated the number of bacteria associated with the epithelium at different time points following bladder contamination using standard colony counts of homogenized bladders. JTV-519 free base A significant drop in bacterial numbers (approximately 90 %) following bladder contamination occurred between 6 and 12 h post-infection (Figure 1C), assisting the notion that BEC loss is a powerful mechanism.