NLRP3 Mediates NF-κB Activation and Cytokine Induction in Microbially Induced and Sterile Inflammation

Nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain containing 3 (NLRP3) has recently emerged as a central regulator of innate immunity and inflammation in response to both sterile inflammatory and microbial invasion signals. Although its ability to drive proteolytic procaspase-1 processing has drawn more attention, NLPR3 can also activate NF-κB. To clarify the physiological relevance of this latter function, we examined the effect of NLRP3 on NF-κB activation and cytokine induction in RNA-interference-based NLRP3-knockdown cell lines generated from the human monocytic cell line THP-1. Knocking down NLRP3 reduced NF-κB activation and cytokine induction in the early stages of Staphylococcus aureus infection. Expression of cytokine genes induced by Staphylococcus aureus was not inhibited by a caspase-1 inhibitor, and did not occur through an autocrine mechanism in response to newly synthesized cytokines. We also demonstrated that NLRP3 could activate NF-κB and induce cytokines in response to sterile signals, monosodium urate crystals and aluminum adjuvant. Thus, NLRP3 mediates NF-κB activation in both sterile and microbially induced inflammation. Our findings show that not only does NLRP3 activate caspase-1 post-translationally, but it also induces multiple cytokine genes in the innate immune system.

For macrophages to produce mature IL-1β, two signals are required. The first (signal 1) activates NF-κB and induces pro-IL-1β synthesis, and the second (signal 2) activates caspase-1 and induces the proteolytic maturation of pro-IL-1β. Since NF-κB activation in response to PAMP stimulation and bacterial infection is normal in macrophages from NLRP3 −/− or ASC −/− mice [12,13], NLRP3 has been considered dispensable for signal 1 in mice. While the TLR pathway is generally believed to mediate signal 1 in the case of pathogen-stimulated macrophages, it is not clear how this signal is mediated in sterile inflammation.
Genetic reconstitution experiments using HEK293 cells have shown that human NLRP3 can activate NF-κB [23,24] in cooperation with ASC. ASC-deficient human monocytic THP-1 cells show defective NF-κB activation and cytokine production in response to Porphyromonas gingivalis infection [25]. However, the role of human NLRP3 in activating NF-κB, especially under sterile inflammatory conditions, has not been examined.
To address human NLRP3's role in the NF-κB signaling pathway, we generated THP-1-derived NLRP3-knockdown cells and demonstrated that NLRP3 mediates NF-κB activation and cytokine gene induction in a caspase-1 independent manner. More importantly, NLRP3 could activate NF-κB and induce cytokines following stimulation by monosodium urate (MSU) crystals and aluminum adjuvant, sterile activators for NLRP3. These results suggest that NLRP3 is important for NF-κB activation, especially in sterile inflammatory diseases.

Generation of micro RNA-based knockdown cell lines under the control of a Tet-on system
Expression cassettes for artificial micro RNAs (miRNAs) targeting NLRP3, ASC, and MyD88 and for negative control miRNAs were generated using the BLOCK-iT Pol II miR RNAi Expression Vector Kit (Life Technologies, Carlsbad, CA, USA) and the following target sequences: for NLRP3, 5 0 -TCCTGAATCAGACTGAAGGCT-3 0 ; for ASC, 5 0 -TAGGTCTCCAGGTAGAAG CTG-3 0 ; and for MyD88, 5 0 -TCTCCAAGTACTCAAAGTCCA-3 0 . The DNA fragment containing the microRNA expression cassette was cloned into a Tet-on lentiviral vector pLVCT-tTRKRAB (Addgene, Cambridge, MA, USA) and transferred into HEK293FT packaging cells using the packaging vectors pMD2G and psPAX2. Recombinant lentiviruses collected from the culture supernatant were used to transduce THP-1 cells. We used vector-coded GFP to select THP-1 cells that expressed pLVCT-tTRKRAB-derived miRNA. The collected cells were cultured without Dox or treated with Dox for 5 d to induce miRNA expression.

Generation of short hairpin (sh) RNA-based knockdown cell lines
The target sequence for NLRP3 (5 0 -GCTGGAATTGTTCTACTGTTT-3 0 ) and a negative control sequence (5 0 -CCTAAGGTTAAGTCGCCCTCG-3 0 ) were used to generate oligonucleotide pairs that were inserted into the pLKO.1 TRC cloning vector (Addgene) to produce lentiviral vectors that were used to transduce THP-1 cells. THP-1 cells stably expressing shRNA were selected by puromycin resistance for 2 weeks, and these cells were used for experiments.

Cell culture and infection with Staphylococcus aureus (S. aureus)
The THP-1 cell line (ATCC TIB-202) and its derivatives expressing miRNA or shRNA were cultured in RPMI 1640 medium supplemented with 10% FCS. To induce IL-1β release, 1x10 5 cells were primed with LPS (20 ng/ml) for 12 h and then with ATP (3 mM) for 1 h. Unprimed THP-1 and THP-derived cells were used for bacterial-infection and MSU-stimulation experiments. For bacterial-infection experiments, S. aureus (strain Smith, kindly provided by Dr, Nakanishi, Kanazawa University, Ishikawa, Japan) was grown in Luria broth until OD 1.9-2.2 at 600 nm, then collected and washed in RPMI 1640 with 10% FCS.
Isolation of primary human monocytes 50 ml of fresh blood from a healthy donor was fractionated using 1.074 g/ml density barrier solution as described [26]. A monocyte-enriched fraction (purity > 80%) was prepared using Pan Monocyte Isolation Kit (Milteny Biotec K.K., Tokyo, Japan).

Statistical analysis
All experiments were done in duplicate or triplicate and repeated at least three times. Error bars indicate standard deviations. Statistical significance of difference between two experimental groups was accessed by two-tailed Student's t-test. P-values are indicated in the text and figures above the two groups. P <0.05 was considered statistically significant.

NF-κB activation and cytokine induction by S. aureus infection were reduced in NLRP3-and ASC-knockdown cell lines
To determine NLRP3's role in activating NF-κB in human monocytes, we generated Dox-inducible, artificial miRNA-based NLRP3-or ASC-knockdown cell lines (miNLRP3 and miASC, respectively) by transducing human monocytic THP-1 cells with Tet-on-regulated lentiviral vectors expressing miRNA against NLRP3 or ASC. Before Dox treatment, the NLRP3 and ASC protein levels in miNLRP3 and miASC cells, respectively, were the same or slightly lower than those in cell lines transduced with negative-control miRNA (miCtrl) (Fig. 1A). After Dox treatment, the NLRP3 and ASC protein levels were markedly reduced in miNLRP3 cells and miASC cells, respectively, but were unaffected in miCtrl cells. In accordance with these results, the IL-1β secretion induced by LPS plus ATP was also reduced in the Dox-treated miNLRP3 and miASC cells (Fig. 1B), further confirming the effectiveness of the target-protein knockdown by this system.
We used the same cell lines to examine NLRP3's role in activating NF-κB and inducing cytokines in response to S. aureus infection [13]. In the Dox-treated NLRP3-and ASC-knockdown cells, not only IL-1β but also TNF-α and IL-8 secretion was dampened through 120 min after S. aureus infection ( Fig. 1C and Fig. A in S1 File), although TNF-α and IL-8 levels in miNLRP3 and miASC cell culture supernatants were comparable in Dox-treated and -untreated cells at a later time point, 180 min after S. aureus infection (Fig. B in S1 File). The TNF-α and IL-1β mRNA induction in response to S. aureus infection was comparable among all cell lines before Dox treatment (Fig. 1D). In contrast, the induction of TNF-α and IL-1β mRNA was markedly reduced in Dox-treated miNLRP3 and miASC cells, but not in miCtrl cells. The effect of NLRP3 knockdown on cytokine induction in early stages of S. aureus infection was reproduced by experiments using shRNA-based NLRP3-knockdown cells (Fig. C in S1 File).
EMSA evaluation of the NF-κB activation after S. aureus infection showed comparable shifted bands corresponding to the NF-κB-DNA complex in the nuclear extracts from all cell lines that were not treated with Dox (Fig. 1E,   miCtrl cells, NF-κB was activated at comparable levels before and after Dox treatment (lanes 2 and 8).
MAPK pathways also contribute to cytokine production in response to bacterial infection. In fact, MAPKs (p38, JNK, and ERK) were activated early in S. aureus infection in the context used in this study (Fig. 1F). However, NLRP3 and ASC knockdown did not affect p38, JNK, or ERK phosphorylation, suggesting that the MAPK pathway is independent of the NLRP3-ASC pathway.
Together, these results indicate that NLRP3 plays an important role in activating NF-κB and inducing cytokine genes in THP-1 cells in response to microbial infection.
TLRs redundantly activate NF-κB at an early stage of S. aureus infection When TLRs recognize bacterial ligands, they activate NF-κB to provoke the release of proinflammatory cytokines. To examine whether the TLR pathway is also involved in activating NF-κB and releasing cytokines in the early stages of infection, we generated MyD88-knockdown cell lines based on a Tet-on system. MyD88 mRNA was slightly reduced in the MyD88 miRNA-introduced (miMyD88) cells even before Dox treatment, and was reduced further by Dox treatment ( Fig. 2A and Fig. 2B). Following S. aureus infection in the Dox-treated MyD88knockdown cells, TNF-α release evaluated by ELISA (Fig. 2C) and NF-κB activation evaluated by EMSA (data not shown) were lower than in Dox-untreated cells. Thus, the NLPR3 and TLR signaling pathways seem to mediate NF-κB activation redundantly or cooperatively in the early stages of S. aureus infection.

NLRP3 mediates NF-κB activation in sterile inflammation
To further investigate the pathophysiological significance of the NLRP3-mediated NF-κB activation, we examined the effect of NLRP3 knockdown on uric acid-induced inflammatory responses. MSU-induced TNF-α and IL-1β mRNA was reduced in the NLRP3-knockdown (shNLRP3) cells compared to control (shCtrl) cells (Fig. 3A). Consistent with this result, MSUinduced TNF-α and IL-1β secretion and intracellular pro-IL-1β accumulation were markedly reduced in shNLRP3 cells ( Fig. 3B and Fig. 3C). The involvement of NLRP3 and ASC in MSUinduced TNF-α and IL-1β expression was confirmed using NLRP3 and ASC-targeting siRNAs, respectively (Fig. D in S1 File). Knocking down MyD88 by miRNA and siRNA reduced the IL-1β-and LPS-induced but not the MSU-induced TNF-α mRNA, indicating that the TLR pathway is not involved in the MSU-induced NF-κB activation ( Fig. 3D and Fig. 3E). These results also rule out the possibility that contaminated LPS was responsible for the MSU-induced cytokine gene expression.
To further confirm the role of NLRP3 in sterile inflammation, we next examined the effect of NLRP3 knockdown on aluminum adjuvant-induced inflammatory responses. For this purpose, PMA-primed shCtrl and shNLRP3 cells were stimulated with aluminum adjuvant. In shNLRP3 cells, the extent of aluminum adjuvant-induced IL-1β mRNA induction was significantly lower than those in shCtrl cells (Fig. 3F). Similar results were obtained in the experiments using unprimed primary human monocytes (Fig. 3G). Together, these results indicate that NLRP3 plays an essential role in the NF-κB activation in sterile inflammation.

No autocrine mechanism is involved in inducing cytokines in early stages of infection
The NLRP3-dependent NF-κB activation and cytokine mRNA induction noted above could have been mediated by quickly released IL-1β through an autocrine mechanism. However, this is unlikely because MyD88 knockdown inhibited the IL-1β-induced but not the MSU-induced TNF-α expression, as described above. To further examine whether the cytokines induced in early infection were mediated by quickly released IL-1β, THP-1 cells were pre-incubated with recombinant IL-1ra, which effectively suppresses the amount of IL-8 released in response to 10 ng/ml recombinant IL-1β, a dose 80-100 times higher than that induced by S. aureus infection (Fig. 4A). IL-1Ra-treated and -untreated cells secreted IL-8 and TNF-α at comparable levels in response to S. aureus infection (Fig. 4B). Consistent with these findings, the caspase-1 inhibitor Ac-YVAD-CMK, which completely blocks IL-1β and IL-18 secretion in response to S. aureus infection (Fig. 4C, right panel and Fig. 4G, left panel), did not affect TNF-α secretion (Fig. 4C,  left panel). Furthermore, while CHX pre-incubation markedly reduced the TNF-α and IL-1β secretion in response to S. aureus infection (Fig. 4D), it did not inhibit TNF-α and IL-1β mRNA induction (Fig. 4E). These results indicate that the mRNA induction seen in early S.  aureus infection is not mediated by an autocrine mechanism involving newly synthesized cytokines. In addition, the MSU-induced TNF-α secretion from THP-1 cells (Fig. 4F, left panel) and aluminum adjuvant-induced TNF-α and IL-1β mRNA expression from human peripheral blood monocytes (Fig. E in S1 File) were not affected by Ac-YVAD-CMK, which dampens MSU-induced IL-1β and IL-18 secretion (Fig. 4F, right panel and Fig. 4G, right panel). These results exclude the possible involvement of caspase-1-dependent secretion of cytokines including IL-1β and IL-18 in the induction of TNF-α.

NLRP3 mediates NF-κB activation downstream of lysosomes
To explore the signaling pathway by which NLRP3 activates NF-κB, we examined a proposed phagolysosome destabilization model for procaspase-1 activation [29,30]. Pre-incubating control and knockdown cells with bafilomycin A1, which neutralizes the lysosomal pH and prevents lysosomal protease maturation, completely blocked the TNF-α mRNA induction and cytokine release (Fig. 5A and Fig. 5B) in the early stages of S. aureus infection. The cytokine release was markedly reduced by pre-incubating control and knockdown cells with the cathepsin B inhibitor CA-074Me (Fig. 5C, CA-074Me). Similarly, the MSU-induced TNF-α secretion was markedly reduced by pre-incubating cells with cytochalasin D, bafilomycin A1, or the NADPH oxidase inhibitor DPI (Fig. 5D and 5E). These results indicate that NLRP3 mediates NF-κB activation downstream of phagolysosome pathways and suggest that, in both sterile and microbially induced immune responses, NF-κB and inflammasomes may be activated concurrently.

Discussion
In this study, we generated RNA-interference-based knockdown cells from the human monocytic THP-1 cell line, and demonstrated that NLRP3 is required for optimal NF-κB activation following S. aureus infection. Previous studies using macrophages from NLRP3 −/− mice demonstrated that NLRP3 is dispensable for NF-κB activation following S. aureus infection [13]; this difference may be species-specific or may result from experimental conditions. In previous studies, mouse macrophages were primed with LPS, which strongly induces TLR4-mediated NF-κB activation, prior to bacterial infection; this would mask NLRP3-mediated NF-κB activation. In our experimental system, NLRP3-NF-κB activation could be detected only during early stages of infection ( Fig. 1 and Fig. B in S1 File), probably because NF-κB is primarily activated through the TLR pathway at later stages of infection.
In sterile inflammatory diseases, signal 1 (for NF-κB activation leading to pro-IL-1β mRNA induction) has been unclear. It has been proposed that TLRs might mediate signal 1 by sensing DAMPs such as high mobility group box-1, hyaluronic acid, and biglycan, which are released from dead cells or extracellular matrix upon tissue injury [2,[31][32][33][34][35]. The data presented here show that NLRP3 is also able to mediate signal 1. Thus, NLRP3 mediates not only signal 2 but also signal 1 to produce IL-1β in sterile inflammation. In primary human monocytes, NLRP3 was also shown to mediate IL-1β mRNA induction. This could explain the spontaneous release of IL-1β by monocytes isolated from CAPS patients carrying gain-of-function NLRP3    [19]. The smaller amounts and slower kinetics of cytokines induced by MSU (Fig. 3) compared with those induced by microbial infection (Fig. 1) may reflect the absence of TLR signaling in response to MSU.
Genetic reconstitution experiments with HEK293 cells have demonstrated that ASC can mediate NF-κB activation. ASC has also been reported to mediate NF-κB and MAPK activation following bacterial infection in THP-1 cells [25,36]. However, one study concluded that NLRP3 is not required to activate MAPKs [36]. Whether ASC mediates the NLRP3-induced NF-κB activation under physiological conditions has not been clarified. Our knockdown experiments indicated that both NLRP3 and ASC are essential for the S. aureus-induced activation of NF-κB but not MAPKs, indicating that ASC mediates the NLRP3-induced NF-κB activation, but NLRP3 and ASC are not involved in MAPK activation under these conditions. The different results in terms of the involvement of ASC in MAPK activation may be due to different infectious agents between this and the previous study.
Our previous genetic reconstitution experiments using HEK293 cells demonstrated that caspase-8 is involved in ASC-mediated NF-κB activation [28,37]. However, neither caspase-8 knockdown nor pretreatment with a caspase-8 inhibitor affected the S. aureus-induced NF-κB activation and cytokine gene expression in THP-1 cells (data not shown). Thus, at least one more mechanism exists for ASC-mediated NF-κB activation under these conditions. In this context, it should be note that NLRP3 and ASC have been reported to interact with MAVS and RIPK2, respectively [38][39][40], that could connect the NLRP3-ASC axis to NF-κB activation pathways. Further study is required to clarify how NLRP3 and ASC activated NF-κB in our experimental conditions. Although NLRP3 has been linked to inflammasome in many microbially and sterile inflammatory responses, inflammasome-independent role for NLRP3 has also been reported [41][42][43]. In our study, pre-treatment with YVAD had no effect on cytokine induction in THP-1 and primary human monocytes (Fig. 4C, Fig. 4F, and Fig. E in S1 File). These suggest a novel caspase-1-independent pathway for NF-κB activation through NLRP3 in immune cells.
In summary, our results indicate that NLRP3 mediates the NF-κB activation in sterile inflammatory and microbially induced immune responses. So far, NLRP3 has been implicated in procaspase-1 activation through inflammasome formation. Our findings significantly broaden NLRP3's physiological importance in innate immunity: NLRP3 not only activates caspase-1 post-translationally, but also induces multiple cytokine genes, including IL1B.
Supporting Information S1 File. Supporting figures.