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. Author manuscript; available in PMC: 2015 Aug 13.
Published in final edited form as: Cell Host Microbe. 2014 Aug 13;16(2):249–256. doi: 10.1016/j.chom.2014.07.002

Non-canonical inflammasome activation of caspase-4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens

Leigh A Knodler 1,2,*, Shauna M Crowley 3, Ho Pan Sham 3, Hyungjun Yang 3, Marie Wrande 1, Caixia Ma 3, Robert K Ernst 4, Olivia Steele-Mortimer 2, Jean Celli 1,2, Bruce A Vallance 3,*
PMCID: PMC4157630  NIHMSID: NIHMS618551  PMID: 25121752

Summary

Inflammasome-mediated host defenses have been extensively studied in innate immune cells. Whether inflammasomes function for innate defense in intestinal epithelial cells, which represent the first line of defense against enteric pathogens, remains unknown. We observed enhanced Salmonella enterica serovar Typhimurium colonization in the intestinal epithelium of caspase-11 deficient mice, but not at systemic sites. In polarized epithelial monolayers, siRNA-mediated depletion of caspase-4, a human orthologue of caspase-11, also led to increased bacterial colonization. Decreased rates of pyroptotic cell death, a host defense mechanism that extrudes S. Typhimurium infected cells from the polarized epithelium, accounted for increased pathogen burdens. The caspase-4 inflammasome also governs activation of the proinflammatory cytokine, interleukin (IL)-18, in response to intracellular (S. Typhimurium) and extracellular (enteropathogenic Escherichia coli) enteric pathogens, via intracellular LPS sensing. Therefore an epithelial cell intrinsic non-canonical inflammasome plays a critical role in antimicrobial defense at the intestinal mucosal surface.

Introduction

Inflammasomes mediate inflammatory host defenses including pyroptosis, a specialized form of cell death, and the cleavage and activation of the proinflammatory cytokines, IL-1β and IL-18 (Ng and Monack, 2013). These responses require the actions of inflammatory caspases, specifically caspase-1, -4, -5 and -12 in humans and caspase-1, -11 and -12 in mice (Ng and Monack, 2013). Caspase-1, the best characterized to date, is cleaved and activated upon recruitment to a multi-protein complex, the inflammasome. Inflammasome assembly is triggered by cytosolic Nod-like receptors (NLRs) that sense microbial- or danger-associated molecular patterns (DAMPs) (Lamkanfi, 2011). Aside from caspase-1-containing inflammasomes, a “non-canonical” inflammasome has been recently described in mouse macrophages which responds to intracellular bacterial lipopolysaccharide (LPS) (Kayagaki et al., 2011). Caspase-11 is activated independently of the LPS receptor, Toll-like receptor 4 (TLR4) (Hagar et al., 2013; Kayagaki et al., 2013); the cytosolic sensor for LPS is unknown. It also remains unclear whether human caspase-4 and/or -5 represent functional orthologues of murine caspase-11.

The primary effectors of inflammasome-mediated control of bacterial infections are believed to be immune cells such as monocytes, macrophages and dendritic cells. Because intestinal epithelial cells (IECs) lie at the host-microbial interface and are an important source of pro-inflammatory cytokines, we hypothesized that inflammasome activation in these cells plays a previously unrecognized role in responding to bacterial infections of the gut. The Gram-negative bacterium Salmonella enterica serovar Typhimurium (S. Typhimurium) infects IECs of several mammalian species, including humans (Santos et al., 2001), causing gastroenteritis. We previously showed that S. Typhimurium infected IECs undergo pyroptosis and release IL-18 (Knodler et al., 2010). Using S. Typhimurium as a model enteric pathogen, we herein describe how the non-canonical epithelial inflammasome promotes host defense and gut inflammation in response to enteric bacteria.

Results

Intestinal epithelial cells require caspase-4 activity for IL-18 secretion

To determine if S. Typhimurium-induced IL-18 secretion required one or more inflammatory caspases, we tested a panel of irreversible, cell-permeable caspase inhibitors. Polarized human colonic epithelial cells (C2Bbe1) were infected with S. Typhimurium and secretion of two proinflammatory cytokines, IL-18 (Knodler et al., 2010) and IL-8 (Jung et al., 1995), assayed by ELISA. IL-18 requires proteolytic processing by an inflammatory caspase prior to secretion, whereas IL-8 does not (van de Veerdonk et al., 2011). Caspase-1, -4 and -5 inhibitors significantly reduced Salmonella-induced IL-18 secretion (Fig. S1A), implicating inflammatory caspase catalytic activity. None of the inhibitors affected IL-8 secretion (Fig. S1A).

mRNA and protein levels of inflammatory caspases in human epithelial cell lines and primary cells indicated caspase-1, -4 and -5 expression, with caspase-4 being the most abundant (Fig. S2). Using small interfering RNA (siRNA) depletion of caspases (Fig. S3) in polarized monolayers, knockdown of caspase-1 had no effect on Salmonella-induced IL-18 secretion, whereas caspase-4 knockdown significantly reduced it (Fig. 1A). No difference in IL-8 secretion was observed (Fig. S1B), demonstrating the selective impact of inflammatory caspase knockdown towards IL-18.

Figure 1. Caspase-4 is required for IL-18 secretion and processing in human intestinal epithelial cells.

Figure 1

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(A) C2Bbe1 cells were electroporated with siRNA targeting caspase-1 (CASP1), caspase-4 (CASP4), caspase-5 (CASP5), interleukin-18 (IL-18) or a non-targeting control (NT). Polarized monolayers were mock-infected or infected with S. Typhimurium or enteropathogenic E. coli (EPEC). Apical and basolateral culture supernatants were collected at 10 h p.i.and secreted IL-18 determined by ELISA. *p<0.05, significantly different from infected, NT siRNA conditions. (B) C2Bbe1 cells were incubated in the presence of 1 μg S. Typhimurium LPS or nucleofected with a dilution series of LPS. At 16 h post-treatment, cell culture supernatants were assayed for IL-18 (grey bars) and IL-8 (white bars) by ELISA. *p<0.05, significantly different from nucleofection with water. (C) Immunoblots of whole cell lysates (WCL) and supernatants (SN) probed for actin and IL-18. HeLa cells were transfected with the indicated siRNA and infected 48 h later with S. Typhimurium. Samples were collected at 10 h p.i. Representative of three independent experiments. (D) HCT 116 cells were mock-infected or infected with S. Typhimurium and cell culture supernatants assayed for IL-18 by ELISA. (E) HeLa cells were treated with NT or caspase-4 siRNA and mock-infected or infected with mCherry S. Typhimurium. Whole cell lysates were analyzed by immunoblotting with antibodies against caspase-4 and actin (representative of three experiments). IL-18 in culture supernatants was assayed by ELISA (black bars). Caspase activity was measured after incubation with FAM-YVAD-FMK FLICA™ reagent. The number of cells with active caspase-1/4/5 was assessed by fluorescence microscopy (grey bars). Asterisks indicate significantly different data. See also Figure S1, S2, S3.

IL-18 is synthesized as a 23 kDa inactive precursor that requires cleavage by an active inflammatory caspase to obtain its mature 18 kDa form (van de Veerdonk et al., 2011). By immunoblotting, mature IL-18 was secreted upon S. Typhimurium infection of IECs (Fig. 1C). siRNA-mediated knockdown of caspase-4, but not caspase-1 or -5, prevented the processing and release of mature IL-18. Salmonella infection also led to the time-dependent release of IL-18 from HCT 116 cells (Fig. 1D), a colonic epithelial cell line which expresses only inflammatory caspase-4 (Fig. S2), confirming that IL-18 secretion does not require caspase-1 in human IECs.

In mouse macrophages, cytosolic Gram-negative bacteria activate the caspase-11 inflammasome (Aachoui et al., 2013), via intracellular LPS detection (Hagar et al., 2013; Kayagaki et al., 2013). LPS delivered to the cytosol of human IECs triggered the caspase-4 inflammasome; extracellular LPS did not (Fig. 1B). Cytosolic LPS stimulated IL-18, but not IL-8, release in a dose-dependent manner (Fig. 1B), implying that IECs possess a cytosolic LPS sensing pathway. Unexpectedly, infection with extracellular enteropathogenic Escherichia coli (EPEC) also led to a caspase-4 dependent induction of IL-18 release from colonic epithelial cells (Fig. 1A).

Expression of some inflammatory caspases is transcriptionally regulated (Broz et al., 2012; Kayagaki et al., 2011; Lin et al., 2000). However, caspase-4 is highly expressed in human IECs (Fig. S2) and only modestly induced upon infection (Fig. 1E), indicating that its expression does not require inflammatory stimulation. The peak times for caspase-1/4/5 activity and IL-18 secretion were concurrent in infected epithelial cells (Fig. 1E). Moreover, siRNA knockdown demonstrated that almost all secreted IL-18 and the majority of FAM-YVAD-FMK positive cells at 20 hours (h) post-infection (p.i.) was due to caspase-4 activity (Fig. 1E). In support of recent findings (Kobayashi et al., 2013), we conclude that enteric bacterial infection of human IECs progressively activates caspase-4, resulting in IL-18 processing and secretion.

Caspase-11 is required for IL-18 secretion during gut inflammation

To test the in vivo relevance of the epithelial inflammasome in pro-inflammatory cytokine release, we used the Salmonella-induced mouse model of gastroenteritis (Barthel et al., 2003; Miller et al., 1956). Caspase-11 is the murine orthologue of human caspase-4 and -5 (Ng and Monack, 2013)., IL-18 and IL-1β secretion from infected cecal tissues of wild type C57BL/6, Casp11−/− or Casp1−/− Casp11−/− mice was quantified (Fig. 2A). Secreted IL-18 levels were significantly lower in cecal explants from Casp11−/− mice compared to wild type mice. IL-1β release was also detectable, consistent with the presence of various myeloid cells in cecal tissues, but there was no difference between wild type and Casp11−/− mice (Fig. 2A). By contrast, both IL-18 and IL-1β release from Casp1−/− Casp11−/− mouse cecal tissues were significantly reduced compared to wild type mice. Hence, caspase-11 is the predominant inflammatory caspase controlling IL-18 release during intestinal S. Typhimurium infection whereas caspase-1, rather than caspase-11, governs intestinal IL-1β responses.

Figure 2. Caspase-11 is required for IL-18, but not IL-1β, secretion during gut inflammation.

Figure 2

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(A) Streptomycin-pretreated C57BL/6, Casp11−/−, Casp1−/− Casp11−/−, Asc−/− and Nlrp3−/− mice were orally infected with ΔaroAS. Typhimurium (3 × 106 CFU) and cecal tissues collected at 3 days p.i. Tissues were washed and incubated in DMEM media for 6 h, culture supernatants collected and cytokine levels measured by ELISA. Each symbol represents one animal. Median is indicated. Results are from ≥2 independent experiments. *p<0.05; n.s., not significant. (B) Streptomycin-pretreated C57BL/6 mice were orally infected as in (A) and cecal tissues collected at 3 days p.i. The lamina propria was separated from crypts to enrich for mononuclear and intestinal epithelial cells, respectively. Crypts were further incubated in DMEM for 3 h and culture supernatants collected. Protein extracts were analyzed by immunoblotting for pro- and mature forms of caspase-1, -11, IL-18 and IL-1β. Cytokeratin 19 (CK19) is a marker of epithelial cells and actin is a loading control. Lysates from two representative mice are shown.

Pro-IL-1β and pro-IL-18 processing upon pathogen activation of the caspase-11 inflammasome in mouse macrophages requires the Nod-like receptor family member, NLRP3, and the adaptor apoptosis-associated speck-like protein (ASC/PYCARD) (Kayagaki et al., 2011). In ASC- and NLRP3-deficient mice, IL-18 and IL-1β release were both significantly reduced from cecal explants compared to wild type mice (Fig. 2A), implicating ASC and NLRP3 in the release of these pro-inflammatory cytokines during Salmonella-induced gastroenteritis.

To determine the cellular origin of the secreted IL-1β and IL-18, whole crypts, enriched for IECs, were separated from the underlying lamina propria of cecal tissues from infected wild type mice. Higher levels of mature caspase-11 and IL-18 were present in the crypt fraction compared to the lamina propria. Mature IL-18, but not IL-1β, was also detected in crypt supernatants (Fig. 2B). By contrast, expression of mature IL-1β and caspase-1 was comparatively increased in the lamina propria (Fig. 2B). Therefore IL-18 activation and secretion primarily occurs in IECs in infected cecal tissues, correlating with mature caspase-11 localization. However, IL-1β production and activation during Salmonella-induced intestinal inflammation is predominantly associated with caspase-1 expression and processing in cells of the lamina propria.

Caspase-4 governs intestinal epithelial shedding rates

S. Typhimurium occupies two distinct niches within human epithelial cells (Knodler et al., 2010; Malik-Kale et al., 2012). Epithelial cells containing cytosolic bacteria die by pyroptosis, ultimately being shed from the monolayer (Knodler et al., 2010). Does caspase-4 promote pyroptotic death of infected IECs? Caspase-4 depletion significantly increased the number of recoverable bacteria in polarized monolayers at 10 h p.i., but caspase-1 and -5 had no effect (Fig. 3B). Conversely, ectopic expression of caspase-4, but not caspase-1 or -5, restricted Salmonella growth (Fig. 3A). Caspase-4 depletion did not affect recoverable bacteria ≤ 7 h p.i. (Fig. 3C), indicating no effect on bacterial internalization or early vacuolar trafficking events. Vacuolar replication of Salmonella was unperturbed by caspase-4 knockdown at 10 h p.i. (Fig. 3D, defined as <40 bacteria/cell, (Malik-Kale et al., 2012)), whereas cytosolic replication was enhanced (≥100 bacteria/cell, (Knodler et al., 2014; Malik-Kale et al., 2012))(Fig. 3D). The increased bacterial burden upon caspase-4 depletion reflects an increased number of IECs containing cytosolic S. Typhimurium, which is independent of mature IL-18 release (Fig. 1B, 3B), implicating pyroptosis instead. Most dying cells contained cytosolic Salmonella (≥100 bacteria/cell) (Fig. 3E) and had a compromised plasma membrane (Figure 3F), regardless of siRNA treatment. However, caspase-4 depletion significantly reduced the proportion of infected, extruding cells with active caspase-1/4/5 i.e. dying by pyroptosis (Fig. 3F). Furthermore, the frequency at which infected cells were shed from epithelial monolayers was decreased in caspase-4 depleted cells (Fig. 3G). Therefore S. Typhimurium induces IEC lysis by more than one mechanism, one of which is caspase-4 dependent and constitutes a key antimicrobial response by IECs to S. Typhimurium infection.

Figure 3. Caspase-4 limits bacterial burdens via epithelial cell shedding.

Figure 3

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(A) C2Bbe1 cells were nucleofected with either pCMV6-XL5 (empty vector control), pCASP1, pCASP4 or pCASP5, infected with S. Typhimurium and solubilized at 8 h p.i. for enumeration of colony forming units (CFU). Means ± SD. (B and C) C2Bbe1 cells were treated with siRNA, polarized on semi-permeable supports and infected with S. Typhimurium. CFU were enumerated at 10 h p.i. (B) or over a time course of infection (C). Means ± SD. *p<0.01 (B); p<0.02 (C), significantly different from infected, NT siRNA conditions. (D, G) C2Bbe1 cells were treated as in B and infected with mCherry S. Typhimurium. Monolayers were fixed at 10 h p.i. (D) or 9 h p.i. (G), immunostained with anti-ZO-1 antibodies and DNA stained with Hoechst 33342. The number of bacteria in each infected cell (D) or the combined number of extruding/extruded infected epithelial cells (G) were scored by fluorescence microscopy. (D) Each dot represents one infected cell. Data are representative of at least three experiments. Percentages indicate the number of infected cells containing ≥100 bacteria/cell. *p<0.05. (G) *p<0.01. (E, F) C2Bbe1 cells were treated as in (B) and infected with S. Typhimurium glmS::mCherry or S. Typhimurium glmS::gfpmut3 for 10 h. (E) Monolayers were fixed and stained with Hoechst 33342 to label epithelial cell nuclei. The number of bacteria in extruding/extruded epithelial cells was scored by fluorescence microscopy. Data was binned into three categories: cells containing 1–19, 20–99 and ≥100 bacteria. (F) Monolayers were incubated with Hoechst 33342 and SYTOX Orange or FAM-YVAD-FMK FLICA. The number of infected, extruding/extruded cells that were SYTOX Orange-positive or FLICA-positive was scored by fluorescence microscopy. *p<0.01.

Caspase-11 restricts bacterial burdens in the intestine

Given that IECs have a non-canonical inflammasome, we assessed the in vivo role of caspase-11 in promoting intestinal host defense against enteric bacteria. Bacterial loads in spleen, liver and mesenteric lymph nodes were comparable between wild type and Casp11−/− mice (Fig. 4A), in agreement with a non-essential role for caspase-11 in controlling S. Typhimurium at systemic sites (Broz et al., 2012). However, Casp11−/− mice had significantly higher pathogen loads in their cecal tissues and lumen (Fig. 4A) and showed a significant reduction in histopathological features of cecal inflammation (Fig. 4B). At early time points, initial colonization of cecal tissues by S. Typhimurium was dramatically different between wild type and Casp11−/− mice (Fig. 4C, 4D), specifically in epithelial cells (Fig. 4E, 4F). While individual bacteria were scattered throughout the cecal epithelium and lamina propria of both mouse strains, numerous crypt epithelial cells containing clusters of >5 S. Typhimurium per cell were evident in Casp11−/− mouse (11.8 epithelial cells/10 high power fields). This colonization phenotype was rarely seen in wild type mice (1.4 epithelial cells/10 high power fields, p<0.01), suggesting that epithelial cell sloughing may be delayed in Casp11−/− mice.

Figure 4. Caspase-11 limits S. Typhimurium burdens in the gut.

Figure 4

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(A) Streptomycin-pretreated C57BL/6 and Casp11−/− mice were orally infected with ΔaroAS. Typhimurium (3 × 106 CFU) and bacterial loads in organs and tissues determined at 7 days p.i. Data are combined from three independent experiments. Each symbol represents one animal (n=13 for BL/6, n=11 for Casp11−/−). Median is indicated. *p<0.05. (B) C57BL/6 wild type and Casp11−/− mice were infected as in (A). Semi-quantitative scoring of inflammation was assessed from hematoxylin and eosin stained cecum sections as described in Experimental Procedures. Each symbol represents one animal (scoring range = 0–17). n=6–16 mice per group. Median is indicated. *p<0.01. (C–F) Streptomycin-pretreated C57BL/6 and Casp11−/− mice were orally infected with GFP-expressing wild type S. Typhimurium (106 CFU) (green). Cecal tissues (day 1 p.i.) were stained with phalloidin to detect actin (red; C and D), anti-cytokeratin 19 (CK19) (E) or anti-epithelial cell adhesion molecule (EpCAM) (F) to detect epithelial cells (red; E and F), and DAPI to detect DNA (blue; C–F). Arrows and arrowheads indicate individual and clusters of bacteria, respectively. Scale bars are 50 μm. (G, H) C57BL/6 and Casp11−/− mice were infected intravenously with wild type GFP-Salmonella (5 × 102 CFU). Gall bladders were collected at day 4 p.i. and stained for CK19 to detect epithelial cells (red) and DAPI to detect DNA (blue). Scale bars are 100 μm. See also Figure S4.

The mouse gall bladder is another site of epithelial cell colonization by S. Typhimurium (Gonzalez-Escobedo and Gunn, 2013; Menendez et al., 2009). Despite no overt difference in the proportion of mice showing evidence of gallbladder infection (8/26 wild type mice, 7/29 Casp11−/− mice), wild type and Casp11−/− mice showed histopathological differences (Fig. S4). Gall bladders of wild type mice showed extensive shedding of epithelial cells laden with bacteria (Fig. 4G), and a heavy infiltration of neutrophils (Fig. S4). Gall bladder epithelial cells of Casp11−/− mice were also filled with Salmonella, but relatively few had sloughed into the lumen (Fig. 4H), and there was little evidence of neutrophil infiltration (Fig. S4). Hence, caspase-11-induced epithelial shedding is important for the clearance of enteric bacteria at mucosal sites in vivo.

Discussion

In the gastrointestinal tract, a single layer of columnar epithelial cells separates the non-sterile lumen from the sterile underlying tissues. Historically, these IECs have been primarily regarded as a mechanical barrier against invading pathogens, whereas the underlying lamina propria and lymphoid tissues, rich in professional immune cells, are considered the main immunological responders in the gut to pathogenic challenge (Artis, 2008). However, IECs can distinguish between commensal and pathogenic microbes, and respond accordingly through the release of antimicrobial factors, suggesting they can participate in the regulation of intestinal immune homeostasis. Here we have demonstrated that non-canonical caspase-4 and caspase-11 inflammasomes govern pathogen clearance and inflammation in vitro and in vivo, respectively, invoking a key role for IECs in gut innate immune defense against enteric bacteria.

At present, demonstration of a role for caspase-11 in restricting bacterial pathogen growth in vivo is limited; Casp11−/− mice carry higher loads of L. pneumophila in their lungs compared to wild type mice (Akhter et al., 2012), and succumb to Burkholderia pseudomallei and B. thailandensis infection, whereas wild type mice do not (Aachoui et al., 2013). In both cases, the mechanisms underlying this control are unknown. Here we uncovered a mechanism for non-canonical inflammasome-mediated restriction of pathogen growth in vivo. Delayed shedding of infected epithelial cells undergoing pyroptosis explains Casp11−/− mice carrying higher intestinal S. Typhimurium burdens. Epithelial cell extrusion is important for maintaining gut homeostasis and barrier function (Gu and Rosenblatt, 2012), and accelerated IEC turnover is a hallmark of infection with many enteric pathogens (Laughlin et al., 2014; Wallis et al., 1986; Ritchie et al., 2012; Kang et al., 2001). Non-canonical inflammasome-mediated epithelial cell extrusion may reflect a generalized gut defense mechanism to eliminate infected cells. In support of this concept, we and others (Kobayashi et al., 2013) have found that S. Typhimurium and EPEC both activate the caspase-4 inflammasome in human IECs. Moreover, it was recently shown that S. flexneri antagonizes IEC death via the actions of a type III effector, OspC3, which binds to cleaved caspase-4, thereby preventing its activation, inhibiting IL-18 release and delaying epithelial cell death (Kobayashi et al., 2013). OspC3 does not bind caspase-5 or -11 and the in vivo relevance of caspase-4 inhibition by S. flexneri remains unknown.

The extent to which human caspase-4 and/or -5 are functional orthologues of mouse caspase-11 remains unclear. Tissue expression of caspase-4 is much more widespread than caspase-5 (Lin et al., 2000; Yin et al., 2009), suggesting cell type- or site-specific roles in inflammasome activation and inflammatory responses. Both caspase-4 and -5 are functional inflammasome components; caspase-4 mediates inflammasome activation in keratinocytes (Sollberger et al., 2012), caspase-5 in THP-1 cells (Martinon et al., 2002) and both partially restrict L. pneumophila growth in THP-1 cells (Akhter et al., 2012). Our data indicates that caspase-4, not caspase-5, is required for IL-18 processing and secretion and pyroptotic cell death in human IECs. Caspase-4 is abundant in IECs (Fig. S2), and only a minor increase in pro-caspase-4 is detected upon infection (Fig. 1E). While expression of caspase-11 is transcriptionally regulated by LPS in mouse macrophages and dendritic cells (Broz et al., 2012; Kayagaki et al., 2011), it is constitutively expressed at high levels in the mouse intestine (Kang et al., 2004). The relative abundance of caspase-4 and -11, and their constitutive expression in epithelial cells and the intestine (Fig. S2) (Demon et al., 2014; Kang et al., 2004), might allow for the rapid sensing of enteric pathogens at mucosal sites.

The human epithelial non-canonical inflammasome is broadly responsive to intracellular and extracellular Gram-negative bacteria, a unique feature to date. LPS-containing outer membrane vesicles shed by extracellular enterohemorrhagic E. coli are internalized by colonic epithelial cells (Bielaszewska et al., 2013), potentially explaining how the inflammasome senses such pathogens. In murine macrophages, caspase-1 is required for non-canonical inflammasome-mediated processing of IL-18 and IL-1β (Broz et al., 2012; Kayagaki et al., 2011). Our data indicates that caspase-11 affects IL-18 processing and secretion, but without testing Casp1−/− mice, we cannot rule out that caspase-1 is also required in murine IECs. By contrast, caspase-4 dependent IL-18 processing and secretion appears to be independent of caspase-1 in human IECs. Caspase-4 is able to cleave IL-18 (Fassy et al., 1998), at the same processing site as human caspase-1 (Gu et al., 1997), which might account for our findings. These distinctive features suggest there are mechanistic differences in non-canonical inflammasome activation between myeloid-derived cells and epithelial cells. Defining these features will be important for establishing the effector functions of caspase-4 and caspase-11 in different cell types and hosts.

Collectively, our work reveals a previously undiscovered host immune defense role for the inflammasome within IECs. Our results show that caspase-4 and -11 are triggered by enteric bacteria and drive inflammasome-based activation and release of IL-18, as well as pyroptotic epithelial cell death and shedding. Importantly, the actions of these inflammatory caspases limit pathogen colonization of the intestinal epithelium, representing a potent mechanism for anti-microbial host defense at mucosal surfaces.

Experimental Procedures

Bacterial strains

Wild type and ΔaroA Salmonella enterica serovar Typhimurium SL1344 (Hoiseth and Stocker, 1981; Månsson et al., 2012), wild type SL1344 harboring pFPV-mCherry or pFPV25.1 for constitutive expression of mCherry or GFP, respectively (Drecktrah et al., 2008; Valdivia and Falkow, 1996) and EPEC O127:H6 wild type strain E2348/69 have been described previously (Levine et al., 1978). SL1344 glmS∷mCherry and glmS∷gfpmut3 were created by site-specific insertion of mCherry (codon-optimized for S. Typhimurium) or gfpmut3 at the attTn7 site of the SL1344 chromosome using pGP-Tn7-Cm (Crépin et al., 2012). Expression of the fluorescent proteins is under the control of the Ptrc promoter, which was amplified from pJC125 (Myeni et al., 2013).

Cell culture

All cell lines were purchased from American Type Culture Collection (ATCC). Caco-2 C2Bbe1 colorectal adenocarcinoma (ATCC CRL-2012), HCT-8 ileocecal colorectal adenocarcinoma (CCL-244), HCT 116 colorectal carcinoma (CCL-247), HeLa cervical adenocarcinoma (CCL-2), HT-29 colorectal adenocarcinoma (HTB-38), HuTu 80 duodenal adenocarcinoma (HTB-40), SW480 colorectal adenocarcinoma (CCL-228) and THP-1 monocytes (TIB-202) were grown as recommended by ATCC. THP-1 monocytes were differentiated with 200 nM phorbol myristic acid (PMA) for 24 h. For polarization, C2Bbe1 cells were grown on collagen-coated cell culture inserts (1 μm pore size, BD Falcon) in 24-well plates for 3 days as described (Knodler et al., 2010). Monolayers with a transepithelial electrical resistance of ≥250 Ω.cm2 were used for infections.

siRNA knockdowns

C2Bbe1 cells (5×105 cells in 20 μl) were transfected in Nucleocuvette™ Strips (Lonza) with 2 μM siRNA using Nucleofector™ solution SE (Lonza) with an Amaxa™ 4D-Nucleofector (program CM-138), then transferred to collagen-coated permeable cell culture inserts for 72 h prior to infection. HeLa cells were seeded in 6-well plates at 1.2×105 cells/well and transfected with DharmaFECT1 reagent (Thermo Scientific) and 25 nM siRNA for 48 h prior to infection. ON-TARGETplus SMARTpool siRNA directed against human IL-18, caspase-1, -4, -5 and a non-targeting pool were from Dharmacon (Thermo Scientific).

Nucleofection of plasmid DNA and LPS

Plasmids encoding human caspase-1, -4 and -5 were purchased from OriGene. Endotoxin-free plasmids were prepared with the Nucleobond Xtra Midi Plus EF kit according to the manufacturer's instructions (Macherey-Nagel). C2Bbe1 cells (4×105 cells in 20 μl Nucleofector™ solution SE) were nucleofected with 1 μg plasmid DNA as described above, then divided between two wells in a collagen-coated 24-well plate for 48 h prior to infection. Alternatively, cells were nucleofected with a dilution series of S. Typhimurium LPS (3 ng-100 ng in cell culture grade water, Corning Cellgro), then divided between two wells in a collagen-coated 24-well plate for 16 h prior to collection of cell-free supernatants.

Bacterial infections

Infection conditions for S. Typhimurium have been described previously (Knodler et al., 2010). For EPEC infections, bacteria were grown static overnight at 37°C in 3 ml Luria-Bertani Miller (LB-Miller) broth. An aliquot (100 μl) of overnight culture was used to inoculate 5 ml Dulbecco's modified Eagle medium (DMEM) and growth continued, statically, at 37°C in 10% CO2 for 3 h. Cells were infected with 1 μl EPEC subculture for 2.5 h at 37°C. Non-adherent bacteria were removed by washing eight times with Hank's balanced salt solution (HBSS) and incubations continued in growth media containing 2 μg/ml gentamicin.

Mouse strains and infections

Casp11−/− and Casp1−/− Casp11−/− mice were obtained from Genentech (Kayagaki et al., 2011). Asc −/− and Nlrp3 −/− mice were obtained from Dr Daniel Muruve (University of Calgary). Nlrp3−/− mice originated from the Department of Biochemistry and the Institute of Arthritis Research, University of Lausanne. C57BL/6 wild-type and knockout mice (8–12 weeks old) were bred under specific pathogen-free conditions at the Child and Family Research Institute. For oral infections, mice were gavaged with streptomycin (100mg/kg) one day before infection,then orally gavaged with an overnight LB culture containing ~2.5 × 106 CFU of wild type or ΔaroA S. Typhimurium SL1344 (StrepR, in some cases carrying pFPV25.1) and sacrificed at specified times p.i. (Månsson et al., 2012). For gallbladder infections, mice were intravenously injected with ~500 CFU of wild type S. Typhimurium SL1344 (in some cases carrying pFPV25.1). For GFP-Salmonella infections, mice were given daily intraperitoneal injections of carbenicillin (100mg/kg), beginning at 30 min before the infection, to maintain the pFPV25.1 plasmid. All mouse experiments were performed according to protocols approved by the University of British Columbia's Animal Care Committee and in direct accordance with the Canadian Council on Animal Care (CCAC) guidelines.

Tissue collection, pathology scoring and bacterial counts

Tissue collection and bacterial counts were as described previously (Knodler et al., 2010; Månsson et al., 2012). In brief, mice were euthanized and the cecum, colon, or gallbladder were collected in 10% neutral buffered formalin (Fisher) or 4% paraformaldehyde (Fisher) for histological analyses. For CFU counts, organs were collected separately, homogenized in PBS pH 7.4 and dilutions plated on LB agar plates containing streptomycin. Cecal pathology was blindly scored by two researchers using hematoxylin-eosin-stained sections as previously described by (Barthel et al., 2003) with the following modification: a score was also given for overall crypt loss within a cross section (0 = none; 1 = up to 25% loss of crypts; 2 = 26–50% loss; 3 = 51–75% loss and 4 = total loss). The cumulative scoring range for cecal inflammation (submucosal edema, PMN infiltration into the lamina propria, goblet cell loss, epithelial integrity and overall crypt loss) was 0–17.

Statistical analysis

The mean ± SD for at least three independent experiments is shown in all figures, unless stated otherwise. p-values were calculated using a one-tailed Student's t-test or analysis of variance (ANOVA) with Dunnett's or Tukey's post-hoc test. A p-value of less than 0.05 was considered to be statistically significant.

Supplementary Material

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Highlights.

  • Enteric pathogens activate an epithelial cell intrinsic non-canonical inflammasome

  • Caspase-4 mediates infected epithelial cell extrusion via induction of pyroptosis

  • Caspase-4 mediates IL-18 cleavage in human epithelial cells

  • Caspase-11 activation controls bacterial burdens in the intestine of mice

Acknowledgements

The authors thank Daniel Muruve and Vishva Dixit for providing mice, Guy Palmer, Mike Konkel, Santanu Bose and Paul Beck for critical reading of this manuscript and Tina Huang, Tregei Starr and Andrew Galbraith for technical assistance. BAV is the Canada Research Chair (Tier 2) in Pediatric Gastroenterology and the CH.I.L.D. Foundation Chair in Pediatric Gastroenterology. This work was supported by start-up funds from the Paul G. Allen School of Global Animal Health (LAK and JC), the Washington State University New Faculty Seed Grant Program (LAK), start-up funds from the University of Maryland - Baltimore (RKE), the Wenner-Gren Foundations and the Swedish Research Council Formas (MW), the Intramural Research Program/DIR/NIAID/NIH (OSM and JC) and grants from Crohn's and Colitis Canada and the Canadian Institutes of Health Research (BAV).

Footnotes

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