Inflammasome-dependent Caspase-1 Activation in Cervical Epithelial Cells Stimulates Growth of the Intracellular Pathogen Chlamydia trachomatis

Inflammasomes have been extensively characterized in monocytes and macrophages, but not in epithelial cells, which are the preferred host cells for many pathogens. Here we show that cervical epithelial cells express a functional inflammasome. Infection of the cells by Chlamydia trachomatis leads to activation of caspase-1, through a process requiring the NOD-like receptor family member NLRP3 and the inflammasome adaptor protein ASC. Secretion of newly synthesized virulence proteins from the chlamydial vacuole through a type III secretion apparatus results in efflux of K⁺ through glibenclamide-sensitive K⁺ channels, which in turn stimulates production of reactive oxygen species. Elevated levels of reactive oxygen species are responsible for NLRP3-dependent caspase-1 activation in the infected cells. In monocytes and macrophages, caspase-1 is involved in processing and secretion of pro-inflammatory cytokines such as interleukin-1β. However, in epithelial cells, which are not known to secrete large quantities of interleukin-1β, caspase-1 has been shown previously to enhance lipid metabolism. Here we show that, in cervical epithelial cells, caspase-1 activation is required for optimal growth of the intracellular chlamydiae.Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States, and it is the leading cause of preventable blindness in the world (1–5). Untreated, C. trachomatis infection in women can cause pelvic inflammatory disease, which can lead to infertility and ectopic pregnancy because of scarring of the ovaries and the Fallopian tubes (6). Infection by the lymphogranuloma venereum (LGV)² strain of C. trachomatis, which has become more common in North America and Europe (7, 8), is characterized by swelling and inflammation of the lymph nodes in the groin (9).Chlamydiae are intracellular pathogens that preferentially infect epithelial mucosa and have a biphasic infection cycle (10). A metabolically inactive form, the elementary body, infects the epithelial host cells through entry vesicles that avoid fusion with host cell lysosomes and develop into a membrane-bound inclusion (11–13). Despite their intravacuolar localization, chlamydiae are still able to acquire nutrients from the host cell and interact with host-cell signaling pathways (13–23). Within a few hours, the elementary bodies differentiate into larger, metabolically active reticulate bodies, which proliferate but are noninfectious. Depending on the strain of C. trachomatis, the reticulate bodies transform back into elementary bodies after 1–3 days and are released into the extracellular medium to infect other cells (11, 24, 25). Chlamydial species possess a type III secretion (T3S) system that secretes bacterial virulence factors into host cell cytosol and may control interactions between the inclusion and host-cell compartments (26).Long before the adaptive immune response is activated, infected epithelial cells produce proinflammatory cytokines and chemokines, including interleukin (IL)-6, IL-8, and granulocyte-macrophage colony-stimulating factor (27), which recruit neutrophils to the site of infection and activate other immune effector cells. However, in many cases the immune system fails to clear the infection, and the chronic release of cytokines becomes a major contributor to the scarring and damage associated with the infection (28–30).The innate immune response during C. trachomatis infection is initiated by chlamydial pathogen-associated molecular patterns, including lipopolysaccharides, which bind to pattern recognition receptors such as Toll-like receptors and cytosolic NOD-like receptors (NLRs), ultimately promoting pro-inflammatory cytokine gene expression and secretion of the cytokine proteins (31–37). However, secretion of the key pro-inflammatory cytokine IL-1β is tightly regulated (38). First, pro-IL-1β is produced following activation of pattern recognition receptor, and the precursor is then cleaved into the mature form by the pro-inflammatory cysteine protease, caspase-1 (also known as interleukin-1 converting enzyme or ICE). The mechanism by which caspase-1 is activated in response to infection or tissue damage was found to be modulated by a macromolecular protein complex termed the “inflammasome,” which consists of an NLR family member, an adaptor protein (apoptosis-associated speck-like protein containing a caspase activation recruitment domain or ASC), and an inactive caspase-1 precursor (pro-caspase-1) (39, 40). Previous studies demonstrated that IL-1β is produced in response to chlamydial infection in dendritic cells, macrophages, and monocytes (41–44). Moreover, C. trachomatis or Chlamydia caviae infection activates caspase-1 in epithelial cells or monocytes (43, 45, 46). However, whether caspase-1 activation during chlamydial infection requires the formation of an inflammasome remains unclear.Previous studies have shown that different pathogens can cause inflammasome-mediated caspase-1 activation in macrophages and monocytes (47). However, epithelial cells lining mucosal surfaces are not only the preferred target for chlamydial infection and other intracellular pathogens but also play an important role in early host immune response to infection by secreting proinflammatory cytokines and chemokines (27). Although epithelial cells are not known to secrete large amounts of IL-1β, inflammasome-dependent caspase-1 activation in epithelial cells is known to contribute to lipid metabolism and membrane regeneration in epithelial cells damaged by the membrane-disrupting toxin, aerolysin (48). As lipids are sorted from the Golgi apparatus to the chlamydial inclusion (13, 15, 49), we therefore investigated whether C. trachomatis induces caspase-1 activation in epithelial cells via the assembly of an inflammasome. We demonstrated that C. trachomatis-induced caspase-1 activation is mediated by an inflammasome containing the NLR member, NLRP3. Several studies have demonstrated the involvement of T3S apparatus in inflammasome-mediated caspase-1 activation by different pathogens in macrophages and monocytes (50–56). Therefore, we further investigated the mechanism by which C. trachomatis triggers the formation of the NLRP3 inflammasome. Our results showed that metabolically active chlamydiae, relying on their T3S apparatus, cause K⁺ efflux, which in turn leads to formation of reactive oxygen species (ROS) and ultimately NLRP3-dependent caspase-1 activation. Epithelial cells do not typically secrete large amounts of IL-1β; instead, caspase-1 activation in cervical epithelial cells contributes to development of the chlamydial inclusion.