Abstract
Inflammasome NLRC4 (NLR family CARD domain containing 4) can protect mucosal barriers such as intestine from invading bacterial pathogens. However, it was incompletely clear how NLRC4 was activated in intestinal epithelial cells. In this study, we demonstrated that LNCGM1082 could mediate the activation of NLRC4 via binding NLRC4 with protein kinase C (PKC)δ. LNCGM1082 knockout (KO) mice had reduced resistance against Salmonella Typhimurium infection, as well as impaired expulsion of infected gut epithelial cells and release of IL-18 upon exposure to S. Typhimurium. Similar to NLRC4 KO and PKCδ knockdown gut organoids, there also was impaired expulsion of gut epithelial cells and release of IL-18 in LNCGM1082 KO gut organoids. Furthermore, there also was reduced activation of caspase-1 and caspase-8 in these LNCGM1082 KO, NLRC4 KO, and PKCδ knockdown gut organoids upon exposure to S. Typhimurium. Our results show that LNCGM1082 in the ICEs plays a critical role in mediating activation of NLRC4 through binding NLRC4 and PKCδ and promoting expulsion of infected epithelial cells and release of IL-18 upon exposure to bacteria such as S. Typhimurium.
Introduction
The intestinal epithelium, a single layer of columnar cells, lines the luminal surface of the intestinal mucosa. It provides a critical barrier to harmful intraluminal entities, including foreign Ags, microorganisms, and their toxin. Therefore, intestinal epithelial cells (IECs) are capable of directly recognizing microbial components through pattern recognition receptors such as inflammasome NLRC4 (NLR family CARD domain containing 4), which is highly expressed in IECs (1, 2). IECs can prevent pathogen access to mucosal tissue and reduce subsequent pathogen dissemination (3). These inflammasomes contribute to inflammatory outcomes by promoting caspase-1– and caspase-8–mediated maturation of cytokine IL-18 and gasdermin D, which can form pores in the cell membrane, leading to pyroptosis (4, 5) or expulsion of infected gut epithelial cells (6). Activation of NLRC4 inflammasomes includes a trigger (e.g., cytosolic flagellin), sensor (NLR apoptosis inhibitory protein [NAIP]), nucleator (NLRC4), adaptor (apoptosis-associated speck-like protein containing a CARD [ASC]), and effector (caspase-1) (7). NLRC4 and NAIP can form a single inflammasome cooperatively as sensor (NAIP) and adaptor (NLRC4) proteins. C57BL/6 mice encode four functional Naip (neuronal apoptosis inhibitory protein) genes 1, 2, 5, and 6, which are highly expressed in IECs (2, 8). The individual NAIPs can act as receptors for different bacterial ligands. NAIP1 and NAIP2 recognize T3SS (type III secretion system) needle and rod from bacteria such as Salmonella Typhimurium, whereas both NAIP5 and NAIP6 recognize flagellin (9). Upon ligand binding, NAIP changes its conformation, allowing oligomerization with NLRC4. Every fully formed oligomer consists of 1 NAIP and 9–11 NLRC4 monomers (10, 11). Activity of NLRC4 inflammasomes not only relies on sensing bacterial components in the cytosol (12), but also phosphorylation (13–16) and ubiquitination (17). However, it was incompletely clear how NLRC4 was activated in the IECs such as that NLRC4 could be phosphorylated by protein kinase C (PKC)δ (13–16), but it was unclear how PKCδ comes close to NLRC4 to phosphorylate NLRC4.
Long noncoding RNAs (lncRNAs), which are not translated into proteins, can modulate transcription, epigenetic modifications, protein/RNA stability, translation, and posttranslational modifications by interacting with DNA, RNAs, and/or proteins (18). The overexpression, deficiency, or mutation of lncRNAs was related to numerous human diseases (19). LncRNAs such as Linc-Cox2, LincRNA-EPS, or Lnc13 have been implicated in immune modulation downstream of pathogen sensors (20). Notably, lncRNAs also serve as scaffolds in the cytoplasm to nucleate complex networks (21), and they directly interact with signaling receptors (22). In this study, we found that LNCGM1082 can bind with NLRC4 and PKCδ in the IECs to cause phosphorylation of the NLRC4, which plays a critical role in the activation of NLRC4.
Materials and Methods
Reagents
Anti-NLRC4 Ab, anti–phospho-NLRC4 Ab, anti-LGR5 Ab, V5 Tag mAb, and DYKDDDDK Tag mAb were from Thermo Fisher Scientific. Anti-PKCδ Ab (Proteintech), anti–IL-18 Ab (Abcam), anti-cleaved caspase-1 Ab, and anti-cleaved caspase-8 Ab (Cell Signaling Technology), and β-actin Ab (Santa Cruz Biotechnology) were purchased. HiPerFect transfection reagent (Qiagen), flagellin (AdipoGen Life Sciences), Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific), TRIzol (Life Technologies), Percoll (Solarbio Life Sciences), gentle cell dissociation and IntestiCult organoid growth medium (STEMCELL Technologies), a pcDNA3.1/V5-His TOPO TA expression kit (Invitrogen), Pierce magnetic RNA-protein pull-down kits (Invitrogen and Thermo Fisher Scientific), and a DIG Northern starter kit (Roche) were purchased. Oligonucleotides are listed in Supplemental Table I.
Mice
LNCGM1082-deficient mice on a C57BL/6J background were generated by the Model Animal Research Center of Nanjing University (Nanjing, China) using the CRISPR-Cas9 system. NLRC4−/− mice were from Prof. F. Shao (National Institute of Biological Sciences, Beijing, China). C57BL/6 mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). All mice were maintained under specific pathogen-free conditions in the Animal Center of Nankai University. All animal experiments were carried out in accordance with the Nankai University Guide for the Care and Use of Laboratory Animals.
Mouse models
S. Typhimurium (SL1344, streptomycin-resistant) infection was performed according to the previous method (23). Briefly, mice were withdrawn from water and food for 4 h before oral gavage treatment and treated by gavage with 25 mg of streptomycin. At 24 h after streptomycin treatment, mice were withdrawn from water and food again and then infected by infusing GFP-labeled S. Typhimurium (GFP-S. Typhimurium; 1 × 107 CFU). Mice were sacrificed on day 4. Representative cecum, liver, and spleen tissues were embedded in OCT compound and frozen over liquid nitrogen for staining. GFP-S. Typhimurium was generated using GFP-expressing vectors and cultured in MacConkey agar liquid medium at 37°C with shaking at 200 rpm overnight, and stored in 25% glycerol PBS frozen stocks and used directly for experiments.
In vitro–cultured and –stimulated organoids
Gut organoid cultures were prepared according to reported methods (24). Briefly, crypts were incubated for 15 min at room temperature in gentle cell dissociation reagent (STEMCELL Technologies). Then, crypts were passed through a 70-μm cell strainer (Biosharp Life Sciences, BS-70-XBS) and collected by centrifugation at 100 × g for 5 min. The crypts were embedded with Matrigel (Corning) and plated, after which the plate was carefully transferred to a 37°C incubator and incubated at 37°C for 10 min to allow domes to polymerization. After polymerization of Matrigel, the crypts were added into IntestiCult medium (STEMCELL Technologies) and the plate was placed in an incubator at 37°C and 5% carbon dioxide. The culture medium was fully exchanged three times per week.
MicroRNA transfection
For stimulation on in vitro–cultured organoids, gut organoids were first cultured and transfected with microRNAs using HiPerFect transfection reagent according to the manufacturer’s instructions. A short hairpin RNA target sequence was chosen by BLOCK-iT RNAi designer (Invitrogen) and/or by i-Score Designer 38.
Ex vivo stimulation
For in vitro stimulation of IECs, IECs from mice were harvested, washed, and incubated with or without 1–2 × 108 S. Typhimurium in DMEM for 2 h after infusion. Total cell lysates and supernatants were analyzed by immunoblotting.
For stimulation on gut organoids, the gut organoids were removed from Matrigel with Dispase (1 mg/ml) for 30 min at 37°C and then suspensions of gut organoids were prepared following dissociation of the cells by pipetting with a P1000 pipet and passing the cells through a 40-mm cell strainer. The cells were pelleted for 5 min at 400 × g, suspended in 100 ml of complete medium, and then stimulated with S. Typhimurium (1 × 107 CFU/ml) for 2 h. Total cell lysates and supernatants were analyzed by immunoblotting.
For inflammasome activation, the gut organoids were primed with LPS (2 μg/ml) for 4 h, and subsequently DOTAP-transfected flagellin (5 μg/ml) for 2 h to activate NLRC4 inflammasome. The cell lysates and supernatants were analyzed by immunoblotting.
RNA extraction and quantitative RT-PCR
Total RNA was extracted from the cells, tissues, and organs using TRIzol reagent according to our previously reported method (25). The fold changes were calculated using the ΔΔCt method according to the manufacturer’s instructions (Applied Biosystems). All of the reactions were run in triplicate.
Northern blot
For Northern blot, harvested total RNAs were run on 1% agarose formaldehyde gel and then transferred to a Hybond nylon membrane using Trans-Blot SD semi-dry electrophoretic transfer according to our previous method (26, 27).
Western blot
For Western blot, our previous method (25) was used in this study. The signals were checked by autoradiography film when HRP substrate was added to the membranes.
H&E staining
For H&E staining, previously reported methods were used in this experiment (25). Gut tissues were fixed in 4% (w/v) paraformaldehyde-buffered saline and embedded in paraffin, after which 5-µm sections of colon sections were cut and stained with H&E.
Immunostaining and RNA fluorescence in situ hybridization
Immunostaining and RNA fluorescence in situ hybridization (FISH) were performed according to our reported protocol (26, 27).
RNA immunoprecipitation
RNA immunoprecipitation (RIP) was performed according to our previously reported protocol (27, 28). RNA was extracted from protein/RNA complexes on the beads using TRIzol reagent and dissolved in DEPC (diethyl pyrocarbonate)-water and quantified by quantitative PCR.
Immunoprecipitation
Immunoprecipitation (IP) was performed according to our previously method (27, 28). The cells were lysed in IP lysis buffer containing 10% PMSF. Protein A/G magnetic beads were denatured and resolved by SDS-PAGE.
RNA-protein pull-down analyses
RNA-protein pull-down analyses were performed using a Pierce magnetic RNA-protein pull-down kit according to our reported method (27, 28). Briefly, transfected and induced cells were harvested and cell lysates were prepared using IP lysis buffers. LNCGM1082 was transcribed and labeled using a RNA 3′ desthiobiotinylation kit in vitro. The samples were analyzed on a gel.
ELISA
For mouse IL-18 ELISA measurement, the supernatants from in vitro–cultured organoids were collected, and then assays were carried out according to the manufacturer’s instructions (Cloud-Clone).
Statistical analysis
Statistical analyses were performed using a two-tailed Student t test and one-way ANOVA/Bonferroni multiple comparison test. The statistical significance of the survival curves was estimated using the Kaplan–Meier method, and the curves were compared using the generalized Wilcoxon test. These were performed by GraphPad Prism 7 software (GraphPad Software). A 95% confidence interval was considered significant and defined as follows: *p < 0.05, **p < 0.01, ***p < 0.001.
Results
Expression of LNCGM1082 in gut epithelial cells
We previously found the expression of LNCGM1082 in myeloid-derived macrophages, which played a critical role in mediating activation of NLRC4 upon exposure to its ligands (29). In this study, we investigated whether LNCGM1082 was also expressed in IECs. Fluorescence hybridization showed the expression of LNCGM1082 in the IECs (Fig. 1A). To further determine the expression of LNCGM1082 in gut epithelial cells, we next set up the gut organoid culture in vitro (Fig. 1B) and found that epithelial cells of gut organoids could indeed express this lncRNA (Fig. 1C). LNCGM1082 expression in both IECs and gut organoids also was further shown by Northern blot (Fig. 1D). As previously reported (24, 30), small intestinal crypts isolated from mice were embedded in Matrigel and cultured in the presence of epidermal growth factor, Noggin, and R-spondin-1. LGR5+ stem cells and lysozyme+ Paneth cells could be detected in these gut organoids (Supplemental Fig. 1). Notably, these intestinal organoids also did not show differences in the domains and circumference per organoid or the organoids per crypt in wild-type (WT) and LNCGM1082 knockout (KO) mice, which were generated by us (29) after in vitro culture for 6 d (Fig. 1, Supplemental Fig. 1, and data not shown). There also was not any difference in stem and Paneth cells in in vitro–cultured gut organoids (Supplemental Fig. 1), indicating that LNCGM1082 did not affect the differentiation and proliferation of the enterocytes. LNCGM1082 was not only detected in the enterocytes but also in the Paneth and stem cells (Supplemental Fig. 1). It was not detected in the IECs and gut organoids of LNCGM1082 KO mice (Fig. 1, Supplemental Fig. 1). Taken together, our results show that IECs can express LNCGM1082.
FIGURE 1.
Expression of LNCGM1082 in intestinal epithelial cells.
(A) RNA-FISH of LNCGM1082 in the intestinal epithelial cells in wild-type (WT) and LNCGM1082−/− mice. NC-FAM represents control probes; blue indicates DAPI staining. Scale bars, 40 μm. (B) In vitro–cultured gut organoids under light microscopes. (C) RNA-FISH of LNCGM1082 in the intestinal organoids of WT and LNCGM1082−/− mice. NC-FAM represents control probes; blue indicates DAPI staining. Scale bars, 20 μm. (D) Northern blot of LNCGM1082 in the intestinal organoids of WT and LNCGM1082−/− mice. All experiments were repeated at least three times. Ctr, control probes.
Binding of LNCGM1082 with NLRC4 in gut epithelial cells
NCGM1082 exerts it role in macrophages through binding with NLRC4 (29). Thus, we also examined whether LNCGM1082 could bind with NLRC4 in gut epithelial cells. The gut organoids cultured for 7 d upon exposure to S. Typhimurium, which exhibited the entrance of S. Typhimurium into gut organoid cells (Fig. 2A), were analyzed for binding of LNCGM1082 with NLRC4. IP showed much more phosphorylated NLRC4 in the gut organoids of WT as compared with that of LNCGM1082 KO mice upon exposure to S. Typhimurium or LPS with flagellin, an activator of NLRC4 (Fig. 2B) (7), suggesting that LNCGM1082 played a role in the phosphorylation of NLRC4. The S. Typhimurium infection model is widely used to detect the roles of NLRC4 (31). RIP also showed binding of LNCGM1082 with NLRC4 upon exposure to S. Typhimurium (Fig. 2C). Pull-down experiments further confirmed binding of NLRC4 with LNCGM1082 in NLRC4-V5 plasmid-transfected gut organoids (Fig. 2D). The binding of LNCGM1082 with NLRC4 was further detected via immunostaining in the gut organoids after exposure to S. Typhimurium (Fig. 2E). Notably, NLRC4 KO did not affect the expression of LNCGM1082, and LNCGM1082 KO did also not affect the expression of NLRC4 (Supplemental Fig. 2), indicating that increased binding of NLRC4 and LNCGM1082 did not result from the changes of NLRC4 and LNCGM1082 expression. Taken together, we demonstrate the binding of LNCGM1082 with NLRC4 in gut epithelial cells.
FIGURE 2.
Binding of LNCGM1082 with NLRC4 in gut epithelial cells.
(A) GFP-labeled S. Typhimurium in gut organoids after exposure to GFP-labeled S. Typhimurium for 2 h.; Red arrows point to S. Typhimurium. NC, negative control. (B) IP of pNLRC4 and then Western blotting in the lyses of gut organoids of WT and LNCGM1082−/− mice after exposure to S. Typhimurium or LPS with flagellin for 2 h. (C) RIP of LNCGM1082 in the IP by NLRC4 in lyses of gut organoid lyses. IP of NLRC4 was analyzed by PCR. (D) Pull-down of LNCGM1082 by NLRC4-V5. Lyses of gut organoids transfected NLRC4-V5 vectors were analyzed. (E) Immunostaining of NLRC4 and FISH of LNCGM1082 in gut organoids after exposure to S. Typhimurium for 2 h. All experiments were repeated at least three times. Scale bard, 20 μm.
LNCGM1082 in gut epithelial cells promotes resistance against S. Typhimurium infection
S. Typhimurium is a common foodborne pathogen. The NAIP/NLRC4 inflammasomes in the IECs were responsible for S. Typhimurium restriction (32). NLRC4 inflammasome-deficient mice were more susceptible to orogastric S. Typhimurium infection (33), which suggested NLRC4-dependent restriction to systemic S. Typhimurium spread. To investigate the function of LNCGM1082 in S. Typhimurium infection, we employed a S. Typhimurium infection model. When S. Typhimurium arrives at the apical surface of the gut epithelium, the pathogen could remain in flagellates and express a preformed T3SS to dock, inject bacterial effector proteins, and invade IECs (34). To allow mucosal colonization by S. Typhimurium, the mice were first treated with streptomycin (35). Meanwhile the littermate mice were used as the control (34, 36). Because experimental mice were treated with streptomycin, only LNCGM1082 KO littermate WT mice were used as a control. GFP-S. Typhimurium was infused into WT, LNCGM1082 KO, and NLRC4 KO mice (Fig. 3A). We first detected fluorescence intensity and found that there was more intense fluorescence in the intestines of LNCGM1082 and NLRC4 KO mice (Fig. 3B, 3C, Supplemental Fig. 3). GFP-S. Typhimurium bacteria in feces were much more numerous in LNCGM1082 and NLRC4 KO mice as compared with controls (Fig. 3D). Survival rates in WT mice were significantly higher than those of LNCGM1082 and NLRC4 KO mice. Additionally, LNCGM1082 KO mice had similar survival rates compared with NLRC4 KO mice. At 3 d postinfection with S. Typhimurium, mice began to show clear clinical symptoms, mainly manifested as lethargy, chills, and rough and messy fur. However, LNCGM1082 and NLRC4 KO mice had more serious clinical symptoms than did WT mice. These results suggest that there was reduced bactericidal ability in LNCGM1082 and NLRC4 KO mice. Bacteria can colonize and infect anatomical sites other than the gastrointestinal tract of the mammalian host, such as the lungs, liver, and spleen (6, 37, 38). Indeed, GFP-S. Typhimurium bacteria in spleen and liver of LNCGM1082 and NLRC4 KO were markedly more prevalent than in control mice (Fig. 3E, 3F). Intestinal tissues in LNCGM1082 and NLRC4 KO mice also showed more damage (Fig. 3G). Taken together, LNCGM1082 in gut epithelial cells promotes resistance against S. Typhimurium infection.
FIGURE 3.
LNCGM1082 promotes resistance against S. Typhimurium infection.
(A) Design of experiments for S. Typhimurium infection. (B) Fluorescence of S. Typhimurium in the intestine of WT, LNCGM1082−/−, and NLRC4−/− mice after infusing GFP-labeled S. Typhimurium for 4 d. (C) Fluorescence of S. Typhimurium in isolated intestine of WT, LNCGM1082−/−, and NLRC4 KO mice after infusing GFP-labeled S. Typhimurium. (D) CFU of S. Typhimurium bacteria in the feces of WT, LNCGM1082−/−, and NLRC4−/− mice. (E and F) S. Typhimurium in the spleen (E) and liver (F) of WT, LNCGM1082−/−, and NLRC4−/− mice after infusing GFP-labeled S. Typhimurium. Scale bars, 20 μm. (G) Epithelial gaps of gut tissues of WT, LNCGM1082−/−, and NLRC4−/− mice after infusing GFP-labeled S. Typhimurium. Scale bars, 20 μm. (H) Survival rate of WT, LNCGM1082−/−, and NLRC4−/− mice after infusing GFP-labeled S. Typhimurium. All experiments were repeated at least three times. Male 6- to 8-wk-old mice (n = 6 mice/group) were used for in vivo experiments. Statistical analysis was performed with a one-way ANOVA/Bonferroni multiple comparison test in (C)–(G) and a Wilcoxon test in (H).
Binding of LNCGM1082 with PKCδ in gut epithelial cells
LNCGM1082 can bind with NLRC4. We next sought to determine how LNCGM1082 mediates activation of NLRC4. NLRC4 could be phosphorylated by PKCδ (13, 15, 16). Together, they formed a complex in the macrophages that led to activation of NLRC4 (13, 14). Thus, we investigated whether LNCGM1082 could also mediate NLRC4 with PKCδ together to activate NLRC4 in gut epithelial cells. Previous findings showed that lncRNA MaIL1 could bind to optineurin, a ubiquitin-adapter platforming TBK1 kinase (21). Thus, we first investigated whether PKCδ could be expressed in gut epithelial cells. Data showed the expression of PKCδ in gut epithelial cells (Fig. 4). We next determined whether LNCGM1082 also bound with PKCδ in gut epithelial cells. IP of the lyses of gut organoids using NLRC4 showed increased PKCδ upon exposure to S. Typhimurium (Fig. 4A). RIP also exhibited the binding of LNCGM1082 and PKCδ (Fig. 4B). Furthermore, this binding of LNCGM1082 with PKCδ was confirmed in PKCδ-V5 plasmid-transfected gut organoids (Fig. 4C). Immunostaining also showed the binding of LNCGM1082 with PKCδ in gut organoids (Fig. 4D). Thus, our result demonstrate that LNCGM1082 can bind with PKCδ.
FIGURE 4.
Binding of LNCGM1082 with PKCδ.
(A) Immunoblotting of PKCδ in the IP by NLRC4 in the WT and LNCGM1082−/− gut organoids after exposure to S. Typhimurium for 2 h. (B) RIP of LNCGM1082 in the IP by PKCδ in the WT and LNCGM1082−/− gut organoid lyses after exposure to S. Typhimurium for 2 h. (C) Pull-down of LNCGM1082 by PKCδ-V5. The lyses of gut organoids transfected PKCδ-V5 vectors were analyzed. (D) Immunostaining of PKCδ and FISH of LNCGM1082 in gut organoids after exposure to S. Typhimurium for 2 h. All experiments were repeated at least three times. Scale bars, 20 μm.
LNCGM1082 promotes expulsion of infected IECs
The recognition of S. Typhimurium by NLRC4 in the IECs could lead to extrusion of epithelial cells to restrict S. Typhimurium replication in the intestinal mucosa during the first day of infection (6). Thus, we also observed whether LNCGM1082 could promote the extrusion of epithelial cells. Indeed, similar to NLRC4 KO mice, LNCGM1082 KO mice had reduced extrusion of epithelial cells after S. Typhimurium infection (Fig. 5A, 5B). We also investigated the extrusion of epithelial cells in gut organoids upon exposure to S. Typhimurium. LNCGM1082 KO indeed affected the expulsion in infected gut epithelial cells (Fig. 5C). NLRC4 KO also showed the suppression on the extrusion of infected epithelial cells in thes gut organoids (Fig. 5). Meanwhile, silencing PKCδ also affected the extrusion of infected epithelial cells upon exposure to S. Typhimurium (Fig. 5C). Thus, LNCGM1082 in gut epithelial cells can promote the extrusion of infected epithelial cells upon exposure to S. Typhimurium.
FIGURE 5.
LNCGM1082 promotes expulsion of infected IECs.
(A) H&E staining of gut epithelial cells in WT, LNCGM1082−/−, and NLRC4−/− mice after infusing S. Typhimurium for 9 h. Scale bars, 40 μm. (B) Immunostaining of the gut epithelial cells in WT and LNCGM1082−/− mice after infusing S. Typhimurium for 9 h. Scale bars, 40 μm. (C) Immunostaining in WT, LNCGM1082−/−, NLRC4−/−, and PKCδ KD gut organoids after exposure to S. Typhimurium for 1.5 h. Scale bars, 20 μm. All experiments were repeated at least three times. Statistical analysis was performed with a one-way ANOVA/Bonferroni multiple comparison test. ***p < 0.001. siNC, small interfering RNA negative control.
LNCGM1082 promotes release of IL-18 in gut epithelial cells
Epithelial inflammasome signaling can lead to mature IL-18 secretion to promote diarrhea, and elicit inflammatory pathology in the intestinal mucosa (6). In naive streptomycin pretreated mice, IL-18 was shown to be dispensable for IEC expulsion (38). Thus, we also investigated the effects of LNCGM1082 on the release of IL-18. IL-18 immunostaining showed that there was a decrease in mature IL-18 in LNCGM1082 KO mice (Fig. 6A–D). Similar results were also observed in NLRC4 KO mice (Fig. 6A–D). We next used gut organoids to observe the effects of LNCGM1082 on the production of IL-18 upon exposure to S. Typhimurium infection. Results showed that decreased mature IL-18 was observed in LNCGM1082 KO gut organoids, and also in NLRC4 KO and PKCδ knockdown (KD) gut organoids (Fig. 6E–J). IL-18 is important in eliciting a number of defenses, including NK cell recruitment, IFN-γ production by NK cells, T cells, and intestinal epithelial lymphocytes, as well as perforin-dependent enteropathy (39). Thus, our results show that LNCGM082 can regulate the production of mature IL-18 to promote inflammation.
FIGURE 6.
LNCGM1082 promotes release of IL-18 in IECs.
(A) Fluorescence of mature IL-18 in gut epithelial cells of WT, LNCGM1082−/−, and NLRC4−/− mice after exposure to S. Typhimurium for 2 h. Scale bars, 40 μm. (B) RT-PCR of IL-18 in the gut epithelial cells of WT, LNCGM1082−/−, and NLRC4−/− mice after exposure to S. Typhimurium for 2 h. (C) Immunoblotting of mature IL-18 in IECs of WT, LNCGM1082−/−, and NLRC4−/− mice after exposure to S. Typhimurium for 2 h. (D) ELISA of IL-18 in gut tissues of WT, LNCGM1082−/−, and MLRC4−/− mice after exposure to S. Typhimurium for 2 h. (E and F) RT-PCR (E) and Western blotting (F) of PKCδ after PKCδ KD using small interfering RNA. (G) Immunostaining of IL-18 in WT, LNCGM1082−/−, NLRC4−/−, and PKCδ KD gut organoids after exposure to S. Typhimurium for 2 h. Scale bars, 20 μm. (H) RT-PCR of IL-18 in WT, LNCGM1082−/−, NLRC4−/−, and PKCδ KD gut organoids after exposure to S. Typhimurium for 2 h. (I) Immunoblotting of IL-18 in WT, LNCGM1082−/−, NLRC4−/−, and PKCδ KD gut organoids after exposure to S. Typhimurium for 2 h. (J) ELISA of IL-18 in WT, LNCGM1082−/−, NLRC4−/− and PKCδ KD gut organoids after exposure to S. Typhimurium for 2 h. All experiments were repeated at least three times. The male, 6-8 wk mice (6 mice/group) were used for in vivo experiments. Statistical analysis was performed with a Student t test (mean ± SD). **p < 0.01, ***p < 0.001. NC, negative control; ns, not significant; siNC, small interfering RNA negative control.
LNCGM1082 promotes activation of caspase-1 and caspase-8
The selective activation of NLRC4 inflammasomes in the IECs leads to a coordinated response that includes cell expulsion and cytokine release (6). This is not fully dependent on caspase-1, but also by caspase-8 that is activated by NLRC4 (6). Epithelial NAIP/NLRC4 signaling can activate either caspase-1 or caspase-8, resulting in pyroptosis or apoptosis, respectively (6, 38). Increased S. Typhimurium susceptibility could be observed in epithelial caspase-8–deficient mice at day 3 (40). A recent study further implicated a potential role for caspase-8 in the expulsion of IECs by NLRC4 inflammasome activation. IL-18 production in gut epithelial cells also depends on activated caspase-1 and caspase-8 (6). Thus, we examined the activation of caspase-1 and caspase-8. We first infected S. Typhimurium and then observed the activated caspase-1 and caspase-8. Immunostaining exhibited decreased fragments of caspase-1 and caspase-8 in LNCGM1082 KO, NLRC4 KO, and PKCδ KD gut organoids of mice, indicating reduced activation of caspase-1 and caspase-8 in these KO or KD gut organoids (Fig. 7A). Decreased fragments of caspase-1 and caspase-8 could also be detected in the gut organoids of LNCGM1082 KO, NLRC4 KO, and PKCδ KD gut organoids by immunoblotting (Fig. 7B). Thus, we demonstrate that LNCGM1082 can induce the activation of caspase-1 and -8, which are related to the expulsion of epithelial cells and release of mature IL-18.
FIGURE 7.
LNCGM1082 promotes activation of caspase-1 and caspase-8 in IECs.
(A) Fragments of caspase-1 and caspase-8 in WT, LNCGM1082−/−, NLRC4−/−, and PKCδ KD gut organoids after exposure to S. Typhimurium for 2 h. Scale bars, 20 μm. (B) Immunoblotting of cleaved caspase-1 and caspase-8 in WT, LNCGM1082−/−, NLRC4−/−, and PKCδ KD gut organoids after exposure to S. Typhimurium for 2 h. All experiments were repeated at least three times. Statistical analysis was performed with a Student t test (mean ± SD). ***p < 0.001. ns, not significant.
Discussion
In this study, we found that LNCGM1082 can bind with NLRC4 and PKCδ to activate NLRC4, which can promote the expulsion of infected epithelial cells and the release of mature IL-18 in gut epithelial cells, and defenses against S. Typhimurium infection. Indeed, the responses triggered by the NLRC4 inflammasome are largely protective against bacterial pathogens such as the release of inflammatory mediators and expulsion of infected cell from the host. In addition, the NLRC4 inflammasome has been implicated in the pathogenesis of colorectal cancer, melanoma, glioma, and breast cancer (41–43). Thus, our data have an important consequence not only in understanding the mechanisms for NLRC4 activation but also its potential application for infectious diseases and tumors.
Activation of NLRC4 inflammasomes not only depended on NAIPs for sensing bacterial components in the cytosol (12) but also phosphorylation (13–16). NLRC4 can be phosphorylated by PKCδ (13, 15, 16). However, it was previously not clear how PKCδ comes close to NLRC4 to phosphorylate NLRC4. In this study, we demonstrate that LNCGM1082 can bind with NLRC4 and PKCδ to form a complex in the gut epithelial cells and then lead to the activation of NLRC4.
NLRC4 can sense pathogenic bacteria, including Gram-negative bacteria, namely Salmonella, Legionella, Shigella, and Pseudomonas spp. These bacteria possess flagellin (a T3SS) or T4SS rod proteins that are recognized by the NAIP proteins, constituting unique binding partners of NLRC4 (44, 45). NAIP1 and NAIP2 recognize the T3SS, whereas flagellin is sensed by NAIP5 and NAIP6. Upon ligand binding, NAIP changes its conformation, allowing oligomerization with NLRC4. Formation of this inflammasome can lead to oligomerization of the CARD domain of NLRC4 and subsequent recruitment and activation of caspase-1 and caspase-8. We demonstrate that LNCGM1082 can promote NLRC4 activation. Because every fully formed oligomer consists of 1 NAIP and 9–11 NLRC4 monomers (10), it was still unclear how LNCGM1082 was to bind to the NLRC4 monomer or the NLRC4 oligomer. However, our data show that LNCGM1082 exerts its role only after exposed to S. Typhimurium or its ligands, which can cause the monomer to form the NLRC4 oligomer (44, 45). Thus, it is possible for LNCGM1082 to bind with NLRC4 oligomer.
We demonstrate that LNCGM1082 can bind with NLRC4 and PKCδ in gut epithelial cells. Other lncRNAs have been recently shown to directly interact with signaling receptors (22). Notably, lncRNAs can serve as scaffolds in the cytoplasm to nucleate complex networks of proteins, which function in signaling transduction programs, such as the TLR–TRIF (Toll/IL-1R domain–containing adapter inducing IFN-β) immune pathway (21). LncRNA MaIL1 acts as a critical structural component of the TLR4 immune signal transduction pathway (21). RNA affinity antisense purification–mass spectrometry revealed that MaIL1 could bind to optineurin, a ubiquitin-adapter platforming TBK1 kinase (21). All of these suggest that the interaction of some intracellular kinases with their substrates needs lncRNAs. A similar phenomenon was also observed by others (46–50). For example, lncRNA HULC directly bound to lactate dehydrogenase A (LDHA) and pyruvate kinase M2 (PKM2) to promote phosphorylation (49), lncRNA LINK-A facilitated the recruitment of BRK to the EGFR/GPNMB complex to BRK kinase activation (47), and LNCRNAAK023948 promoted interaction between DHX9 and p85, which could enhance p85 stability and AKT activity (50).
In conclusion, we find that LNCGM1082 plays a critical role in the activation of NLRC4 upon exposure to bacteria such as S. Typhimurium, which can promote expulsion of infected epithelial cells and release of IL-18. Mechanically, we demonstrate that LNCGM1082 can bind NLRC4 and PKCδ in gut epithelial cells to promote phosphorylation of NLRC4.
Supplementary Material
Footnotes
This work was supported by National Natural Science Foundation of China Grants 91842302, 81970488, 81970457, 91629102 and 82174374; Tianjin Science and Technology Commission Grants 20JCQNJC01780 and 18JCZDJC35300; Ministry of Science and Technology Grant 2016YFC1303604; and by the State Key Laboratory of Medicinal Chemical Biology and the Fundamental Research Funds for the Central University, Nankai University Grant 63191724.
The online version of this article contains supplemental material.
- FISH
- fluorescence in situ hybridization
- IEC
- intestinal epithelial cell
- IP
- immunoprecipitation
- KD
- knockdown
- KO
- knockout
- lncRNA
- long noncoding RNA
- NAIP
- NLR apoptosis inhibitory protein
- NLRC4
- NLR family CARD domain containing 4
- PKC
- protein kinase C
- RIP
- RNA immunoprecipitation
- T3SS
- type III secretion system
- WT
- wild-type
Disclosures
The authors have no financial conflicts of interest.
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