Unsaturated fatty acids prevent activation of NLRP3 inflammasome in human monocytes/macrophages.

The NLRP3 inflammasome is involved in many obesity-associated diseases, such as type 2 diabetes, atherosclerosis, and gouty arthritis, through its ability to induce interleukin (IL)-1β release. The molecular link between obesity and inflammasome activation is still unclear, but free fatty acids have been proposed as one triggering event. Here we reported opposite effects of saturated fatty acids (SFAs) compared with unsaturated fatty acids (UFAs) on NLRP3 inflammasome in human monocytes/macrophages. Palmitate and stearate, both SFAs, triggered IL-1β secretion in a caspase-1/ASC/NLRP3-dependent pathway. Unlike SFAs, the UFAs oleate and linoleate did not lead to IL-1β secretion. In addition, they totally prevented the IL-1β release induced by SFAs and, with less efficiency, by a broad range of NLRP3 inducers, including nigericin, alum, and monosodium urate. UFAs did not affect the transcriptional effect of SFAs, suggesting a specific effect on the NLRP3 activation. These results provide a new anti-inflammatory mechanism of UFAs by preventing the activation of the NLRP3 inflammasome and, therefore, IL-1β processing. By this way, UFAs might play a protective role in NLRP3-associated diseases.


Generation of stable THP-1 cell line expressing shRNA
The THP-1 cells stably expressing shRNA were obtained by lentiviral transduction carried out by the GIGA-Viral Vectors Platform (GIGA, Liege, Belgium). In summary, shRNA and promoter sequences were amplifi ed by PCR from commercial plasmids encoding a shRNA against human NLRP3 (Sigma, #TRCN0000427726) or a nontarget sequence (Sigma, #SHC002) and cloned into pHRSin-CSGW plasmid expressing GFP [a kind gift from Dr. Els Veroyen (University of Lyon)]. New lentiviral plasmids were cotransfected with pSPAX2 (Addgene, #12260) and a VSV-G-encoding plasmid ( 16 ) in Lenti-X 293T cells (Clontech, #632180). Viral supernatants were collected, fi ltered, and concentrated 100× by ultracentrifugation. Finally, THP-1 cells were transduced with these lentiviral vectors and GFP-positive cells were sorted by FACS.

esiRNA transfection
Cells were transfected by using the HiPerFect transfection reagent (Qiagen) according to the manufacturer's instructions for suspension cell lines, with minor modifi cations. THP-1 cells (0.2 × 10 5 ) were plated in a 24-well plate in 100 µl of supplemented medium. Transfection complexes were prepared in RPMI 1640 without serum by adding 3 µl of HiPerFect and 400 ng of esiRNA in a fi nal volume of 100 µl and mixed with suspension cells. After 6 h, 400 µl of supplemented medium were added. The next day, differentiation was performed with PMA as previously described. The esiRNAs used were from Sigma: esiPYCARD (#EHU066851) for ASC, and esiNLRP3 (#EHU071121) and esiEGFP (#EHUEGFP) for negative control.
The molecular link between obesity and NLRP3-mediated IL-1 ␤ release is not well established. Free fatty acids (FFAs), usually elevated in plasma of obese people, have been proposed as one triggering event ( 1 ). Recently, the saturated fatty acid (SFA) palmitate demonstrated its ability to activate the NLRP3 infl ammasome in murine macrophages ( 14 ). In the present study, we tested the four most important FFAs present in blood ( 15 ) on human macrophages: two SFAs, palmitate and stearate, and two unsaturated fatty acids (UFAs), oleate and linoleate. We identifi ed stearate as a new physiological NLRP3 inducer, acting with the same effi ciency as palmitate on the caspase-1/ASC/NLRP3 pathway. UFAs, which did not activate the NLRP3 infl ammasome, revealed an unexpected anti-infl ammatory property through preventing NLRP3 infl ammasome activation by SFAs and by various inducers including nigericin, alum, and monosodium urate (MSU).

Preparation of FFA solutions
The palmitic acid (#P0500), stearic acid (#S4751), oleic acid (#O1008), and linoleic acid (#L1376) were purchased from Sigma. A 100 mM stock solution of sodium salt was prepared by dissolving fatty acids in 0.1 M NaOH at 30°C, 37°C, 65°C, and 75°C for linoleic acid, oleic acid, palmitic acid, and stearic acid, respectively. Stock solutions were aliquoted and stored at Ϫ 20°C for less than one year. A 5% fatty acid free, low endotoxin BSA (Sigma, #A8806) solution was prepared in RPMI 1640. The FFA stock solution and the 5% BSA solution were mixed together to obtain a 2.5 mM working solution with FFA:BSA molar ratio at 3.4:1. After pH adjustment, the working solution was fi ltered through a 0.2 µm pore size membrane fi lter, aliquoted, and stored at Ϫ 20°C for less than two months.

Cell culture and treatment
The THP-1 monocytic cell line (ATCC) (authentication in 2011 by DSMZ, Germany) and THP1-XBlue™ cells (InvivoGen) were cultured between 0.5 and 1.0 × 10 6 cells/ml in RPMI 1640 with glutamine supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 U/ml streptomycin and maintained at 37°C under 5% CO 2 atmosphere. Medium and reagents were purchased from Lonza. THP-1 and THP1-XBlue™ cells were differentiated with 100 ng/ml of PMA (Sigma) for 24 h, washed with PBS, and kept one night at rest in fresh supplemented medium before stimulation. Primary monocytes were isolated from human peripheral blood mononuclear cells (PBMCs) by using CD14 MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions. PBMCs were obtained by single-step density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare) from buffy coats (Red Cross, Belgium). Buffy coats were obtained from healthy donors after informed consent, and experiments were approved by the ethical committee of the University of Liege. Primary monocyte purity was evaluated by fl ow cytometry sucrose, 0.1 mM PMSF, 1 mM DTT, 1 mM sodium orthovanadate, 25 mM ␤ -glycerophosphate, 15 mM sodium fl uoride, complete protease inhibitor cocktail, and 4 mM of disuccinimidyl suberate (DSS; Pierce)] was added for 15 min at room temperature under agitation. Cells were scraped and transferred to a microcentrifuge tube. After lysis by shearing 20× through a 21 gauge needle, cells were kept under agitation at room temperature for 20 min. Crosslinking was stopped by addition of Tris, and samples were kept on ice. To remove nuclei, samples were fi ltered through 5 µm pore size membrane fi lter. Crosslinked complexes were precipitated by centrifugation at full speed for 3 min at 4°C, and pellets were resuspended in total phospho lysis buffer before immunoblot analysis.

NF-B/AP-1 activity assay
THP1-XBlue™ cells were differentiated and treated as describe above. After treatment, supernatants were mixed with QUANTI-Blue™ (InvivoGen) according to the manufacturer's instructions to detect alkaline phosphatase activity. Absorbance was measured at 630 nm on the EnSpire 2300 Multilabel Reader.

Statistical analyses
All statistical analyses were carried out using GraphPad Prism 5 for Windows (GraphPad Software, Inc.) and presented as means ± SEM. The Student t -test was performed for simple comparison (two groups) and the ANOVA test followed by Bonferroni post-test for multiple comparisons (more than two groups). Signifi cance is indicated by a symbol.

SFA induce IL-1 ␤ secretion by activating the NLRP3 infl ammasome in human monocytes/macrophages
FFAs are poorly soluble compounds in aqueous solution, and most of them are bound to serum albumin in human physiology ( 18 ). For in vitro experiments, human serum albumin is often substituted by BSA as we did. Among FFAs, palmitate (C16:0) demonstrated its ability to induce IL-1 ␤ secretion in murine macrophages through a caspase-1/ASC/NLRP3-dependent pathway ( 14 ). In human primary monocytes primed with LPS to increase expression of both NLRP3 and pro-IL-1 ␤ ( Fig. 1A , bottom), we showed that stearate (C18:0) was also able to induce IL-1 ␤ release with similar effi ciency as palmitate when compared with vehicle alone (BSA) ( Fig. 1A , top). On the other hand, oleate (C18:1) and linoleate (C18:2) had no effect on IL-1 ␤ secretion, showing opposite effects of SFAs compared with UFAs. Similar fi ndings were observed in MDMs ( Fig. 1B ) and in M1-polarized macrophages ( Fig.  1C ), the predominant phenotype of macrophages present in the adipose tissue from obese. The release of IL-1 ␤ observed after LPS-priming ( Fig.1A ) or M1-polarization ( Fig.1C ) is linked to pro-IL-1 ␤ synthesis. Indeed, monocytes have a constitutive caspase-1 activity ( 19 ), resulting in a background of IL-1 ␤ release related to pro-IL-1 ␤ content.

ELISA
Mature IL-1 ␤ was quantifi ed in supernatants by ELISA with Quantikine for human IL-1 ␤ (R & D Systems) according to the manufacturer's recommendations.

qRT-PCR
Total RNAs were extracted with RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol, followed by DNase treatment (Roche). After quantifi cation by spectrophotometer Nanodrop 1000 (Thermo scientifi c), 500 ng of RNA were reverse-transcribed by using the moloney murine leukemia virus reverse transcriptase (Invitrogen). Obtained cDNA was submitted to qRT-PCR on a LightCycler 480 (Roche Applied Science). Relative gene expression was calculated using the 2 -delta delta C T method ( 17 ) with HPRT1 as housekeeping gene. Results are representative from three independent experiments quantifi ed in triplicate. The following primers were used: IL-1 ␤ -Fw: 5 ′ -CCTTGGGCCTCAAGGAAAA-3 ′ ; IL-1 ␤ -Rv:

ASC dimerization
PMA-differentiated THP-1 cells (10 7 cells/dish) were seeded in 60 cm 2 culture dishes. After 4 h of treatment, cells were washed with PBS and oligoASC buffer [20 mM HEPES-KOH pH 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 1 mM EDTA, 320 mM inhibition repressed it almost completely ( Fig. 2B ). This result suggests a key role for caspase-1 in SFA-induced IL-1 ␤ processing, even if a minor contribution of a second caspase is not excluded.
ASC is an adaptor protein commonly involved in caspase-1 processing. It is composed of two domains mediating protein-protein interactions: the pyrin domain (PYD) and the CARD domain, which is essential for the homotypic interaction with caspase-1. When ASC was knocked down by esiRNA in PMA-differentiated THP-1 cells, a decrease in IL-1 ␤ secretion was observed after SFAs treatment ( Fig. 2C ). ASC can participate in caspase-1 activation through formation of two types of complexes: the pyroptosome and PMA. This classical ( 20,21 ) and highly responsive model allowed us to easily detect large amounts of IL-1 ␤ in supernatant by Western blot and to focus on the maturation process. As in primary monocytes/macrophages, SFAs increased the IL-1 ␤ secretion in PMA-differentiated THP-1 cells in a dose-dependent manner ( Fig. 2A ). The 17 kDa form of IL-1 ␤ observed in Fig. 2A is known to be mainly produced after a proteolytic cleavage by caspase-1 ( 2, 3 ). To investigate caspase-1 involvement, the PMA-differentiated THP-1 cells were treated with SFAs alone or along with a pan-caspase inhibitor (Z-VAD) or with a caspase-1-specifi c inhibitor (Z-YVAD). Inhibition of caspases completely abolished SFA-induced IL-1 ␤ release, while caspase-1-specifi c  induced by SFAs, with equal effi ciency for both oleate and linoleate ( Fig. 3A ). In M1-polarized macrophages, UFAs also decreased SFA-induced IL-1 ␤ release ( Fig. 3B, C ).
The affi nity of long-chain fatty acids for BSA weakly differs according to each FFA ( 22 ). Therefore, if SFAs have more affi nity to BSA than do UFAs, the addition of BSA the infl ammasome. The pyroptosome is produced after dimerization of ASC through its pyrin domain, allowing recruitment and activation of caspase-1 ( 7 ). To examine pyroptosome assembly, PMA-differentiated THP-1 cell extracts were crosslinked with DSS to detect ASC dimers. Compared with LPS at high concentration, a wellcharacterized pyroptosome inducer in PMA-differentiated THP-1 cells ( 7 ), neither palmitate nor stearate led to ASC dimerization ( Fig. 2D ). This indicates that ASC involvement in SFA-induced IL-1 ␤ secretion is not due to pyroptosome formation.
The second complex formed with ASC protein is the infl ammasome. This complex appears by interaction between the PYD domain of ASC and the PYD domain of a second protein, such as NLRP3 or AIM2 ( 3 ). As for pyroptosome, ASC interaction leads to caspase-1 recruitment. Among the infl ammasomes, the NLRP3 infl ammasome is the most extensively studied, and a wide variety of compounds is known to activate it ( 3 ). The knockdown of NLRP3 in PMA-differentiated THP-1 cells by esiRNA led to a strong inhibition of SFA-induced IL-1 ␤ release ( Fig. 2C ). Similar results were obtained in THP-1 cells stably expressing shRNA against NLRP3 ( Fig. 2E ), confi rming that both palmitate and stearate lead to a NLRP3 infl ammasomedependent IL-1 ␤ processing.

UFAs prevent SFA-induced IL-1 ␤ maturation
Unlike SFAs, UFAs possess double bounds that confer their particular properties. As described previously, they cannot activate the NLRP3 infl ammasome compared with SFAs. To determine the impact of UFAs on SFA-induced IL-1 ␤ release, we treated LPS-primed primary monocytes with SFAs alone or in combination with UFAs. Interestingly, the presence of UFAs totally abolished the IL-1 ␤ secretion  protein. To overcome this problem, a different approach was used. As explained previously, the NLRP3 infl ammasome is known to be activated by various compounds ( 3 ). Therefore, we tested UFAs outcomes on the most common NLRP3 activators such as nigericin, ATP, alum crystals and MSU in LPS-primed monocytes ( Fig. 5A , B ) and in PMA-differentiated THP-1 cells ( Fig. 5C, D ). Cotreatment with UFAs signifi cantly inhibited the IL-1 ␤ release induced by SFAs but also by nigericin, alum, and MSU ( Fig. 5B, D ). On the other side, ATP seemed to be insensitive to UFAs after 8 h of cotreatment in both LPS-primed monocytes and PMAdifferentiated THP-1 cells.
NLRP3 is one protein among many others leading to an infl ammasome assembly. To explore other infl ammasomes, we next treated LPS-primed monocytes with NLRC4, AIM2 or NLRP7 activators, respectively fl agellin, poly(dA:dT) or FSL-1 ( Fig. 6A ). UFAs cotreatment had no signifi cant effect on IL-1 ␤ release induced by these compounds ( Fig. 6B ). Taken together, these results suggest a specifi c action on the NLRP3 infl ammasome, probably upstream of infl ammasome assembly.
could mediate the inhibitory effect by decreasing the unbound fraction, which is the active form. To exclude this option, we normalized the BSA concentration to evaluate the specifi c impact of UFAs on palmitate-induced IL-1 ␤ secretion in PMA-differentiated THP-1 cells ( Fig. 3D ). With equal amounts of BSA, the inhibiting effect of UFAs persisted and was obviously dose dependent. Moreover, BSA alone had no effect. Similar results were obtained when cells were stimulated with stearate ( Fig. 3E ). UFAs were effi cient in small concentrations, and the UFA:SFA ratio of 1:2 provided good inhibition. However, a ratio of 1:1 was used for all subsequent experiments.
Processing and secretion are two important regulatory events for IL-1 ␤ release ( 2 ). To identify which step is targeted by UFAs, we assessed the maturation process in PMA-differentiated THP-1 cells. Cotreatment with UFAs strongly reduced the intracellular pro-IL-1 ␤ cleavage ( Fig. 4A ) and the caspase-1 activation ( Fig. 4B ) induced by SFAs. Considering that SFAs activate the NLRP3 infl ammasome, the inhibiting effect of UFAs on IL-1 ␤ maturation probably operates by preventing SFA-induced NLRP3 infl ammasome activation.

UFAs decrease NLRP3 infl ammasome activation by various inducers
The hallmark of NLRP3 infl ammasome formation is the interaction between NLRP3 and ASC. Unfortunately, the low amount of endogenous NLRP3 and the lack of good antibodies against the human form make this interaction diffi cult to observe without over-expressing a tagged NLRP3 Because NLRP3 and IL-1 ␤ are both NF-B-dependent genes ( 8 ), SFAs could likely play a priming role as it was previously described in dendritic cells ( 28 ). Although IL-1 ␤ and NLRP3 genes were already induced by PMA in THP-1 cells, a further induction of NLRP3 and IL-1 ␤ mRNA was observed by qRT-PCR after treatment with SFAs but not with UFAs ( Fig. 7D ), suggesting a "second priming" concomitantly to NLRP3 infl ammasome activation in PMA-differentiated THP-1 cells. ASC and caspase-1 mRNA did not change. This low induction in gene expression is specifi c of the PMA-differentiated THP-1 model, and no further induction of NF-B activation was observed in LPS-primed monocytes (data not shown).
We next examined the impact of UFAs on this further induction of gene expression. Addition of UFAs did not change the phosphorylation of I B ␣ and p65 induced by SFAs in PMA-differentiated THP-1 cells ( Fig. 8A ). No signifi cant effect was observed on IL-1 ␤ ( Fig. 8B ) or NLRP3 ( Fig. 8C ) mRNA levels when UFAs were present. Taken together, these results strongly suggest that i ) SFAs increase little or no gene expression in primed cells, confi rming that the IL-1 ␤ release observed is mainly due to an enhancement in processing; ii ) the decrease in IL-1 ␤ secretion by cotreatment with UFAs is not linked to reduction of transcription but to reduction of IL-1 ␤ processing. DISCUSSION When the immune system is activated, all the resources are mobilized to eradicate the aggressor. At term, the infl ammatory response ends and the immune system comes back to normal. In particular cases, the immune system is ineffective to resolve the trouble and a chronic infl ammation appears. In obesity, an unexplained chronic, lowgrade infl ammation is present ( 29 ) in which SFAs could play a role. As reported here, both SFAs palmitate and stearate activated the NLRP3 infl ammasome and led to IL-1 ␤ secretion. Unlike SFAs, the UFAs oleate and linoleate were unable to activate the NRLP3 infl ammasome, showing opposite effects of SFAs versus UFAs. Our results confi rm in human monocytes/macrophages previous work demonstrating the NLRP3 activation in mouse macrophages by palmitate ( 14 ) and identify stearate as a new inducer. Stearate is the second most important SFA in blood and represents with palmitate 90% of total SFAs ( 15 ), while other SFAs, such as laurate and myristate, are present in very low concentrations. This proinfl ammatory effect of SFAs in vitro corroborates interventional studies describing that SFA-rich diets increase IL-1 ␤ production ( 4, 30 ) as well as also other infl ammatory processes that may be related to IL-1 ␤ ( 31-34 ).
In PMA-differentiated THP-1 cells, we demonstrated a weak induction of IL-1 ␤ and NLRP3 gene expression by SFAs. Even if it was low, this result means that the increase in IL-1 ␤ secretion after SFA treatment can also be, in part, of transcriptional origin. The lack of further induction reported in LPS-primed monocytes is likely inherent to the

UFAs reduce IL-1 ␤ processing but not transcription
Nuclear factor-kappa B (NF-B) family members are critical regulators of gene expression in mammals ( 26 ). These transcription factors bind as dimers to B response elements in diverse gene promoters. Without stimulation, this dimer is sequestered in the cytoplasm by the inhibitory protein I B ␣ . Upon activation of the canonical pathway, I B ␣ is phosphorylated, ubiquitinated, and fi nally submitted to proteasomal degradation allowing the p50/p65 heterodimer translocation to nucleus. Various posttranslational modifi cations are known to change the transcriptional activity of p50/p65, such as the activating phosphorylation in the transactivation domain of p65 ( 26 ). SFAs are known to activate the canonical NF-B pathway in nondifferentiated THP-1 cells ( 27 ). In PMA-differentiated THP1-XBlue cells, a THP-1 cell line stably expressing a reporter gene under the control of NF-B and AP-1 transcription factors, an increase of the transcriptional activity was reported after SFA but not after UFA treatment ( Fig. 7A ). Since IL-1 ␤ also activates the NF-B pathway and to exclude the artifact related to its release, subsequent experiments were performed before IL-1 ␤ secretion. IL-1 ␤ release occurred at around 4 h and became signifi cant at 6 h post-treatment ( Fig. 7B ). After 3 h, SFAs increased phosphorylation of I B ␣ and p65 in PMA-differentiated THP-1 cells ( Fig. 7C ). No increase in phosphorylation was observed after UFA treatment. the NLRP3 infl ammasome activation in macrophages ( 30 ). They also tested non-omega-3 fatty acids, such as oleate (but not linoleate), but they failed to observe an inhibition. This surprising result is likely due to their experimental conditions. Indeed, they used a very low concentration of oleate, lower than the physiological level, to work with a concentration corresponding to omega-3.
UFAs are known to exert a broad range of effects, such as changes in membrane composition ( 41 ), generation of anti-infl ammatory compounds, such as resolvins ( 42 ), or activation of various receptors ( 43 ). G protein-coupled receptor (GPR)40 and GPR120 are both activated by longchain fatty acids. Inhibition of the NLRP3 infl ammasome by omega-3 was described to be dependent on these two receptors through initiation of ␤ -arrestin-2 binding to NLRP3 ( 30 ). Therefore, one explanation could be that oleate and linoleate prevent NLRP3 infl ammasome activation by binding these receptors but with less affi nity than omega-3, explaining the requirement of a higher concentration. A second mechanism could involve the endoplasmic reticulum (ER). UFAs could prevent ER stress induction ( 44,45 ), recently described to be involved in infl ammasome activation ( 20,46 ). In addition, SFAs or tunicamycin, both sensitive to UFA inhibition, are well-characterized ER stress inducers ( 20,(44)(45)(46).
An outstanding issue is the lack of response to UFAs when NLRP3 was activated by ATP. Numerous studies have described contradictory results depending on the activators tested (47)(48)(49). This wide heterogeneity in response to NLRP3 inducers clearly suggests that several pathways are able to trigger the NLRP3 infl ammasome assembly. model used. Indeed, compared with PMA-differentiated THP-1 cells, there is no resting period between the priming and the stimulation with FFAs. This phenomenon is called "tolerance" and the NF-B activation by priming agent leads to a transient unresponsive state in the second stimulation ( 35 ). Therefore, a priming action of SFAs is not excluded in unprimed monocytes, as it was clearly demonstrated in dendritic cells ( 28 ). In our study, primed monocytes were chosen to reduce to a minimum the transcriptional effect of SFAs and to focus on the second signal, the NLRP3 infl ammasome activation.
IL-1 ␤ involvement in type 2 diabetes, gouty arthritis, or atherosclerosis is clear and well documented ( 1 ). It is strongly suggested that the NLRP3 infl ammasome plays a crucial role in the transition between obesity and these diseases ( 36 ). Based on that, new treatments are developed in the hope of avoiding these obesity-associated complications. Treatment with IL-1 ␤ antagonists provided benefi cial outcomes in many diseases, including type 2 diabetes ( 37 ) and gouty arthritis ( 38 ). Similar experiments are being conducted to prevent cardiovascular diseases ( 39 ), with very encouraging preliminary results ( 40 ). In our study, we report an unexpected effect of UFAs on NLRP3 infl ammasome activation. Both oleate and linoleate prevent, with the same effi ciency, the NLRP3-dependent IL-1 ␤ processing induced by SFAs but also by many activators, such as nigericin, alum, or MSU. However, no effect was observed on ATP-mediated NLRP3 infl ammasome activation, which suggests an action upstream of the NLRP3 infl ammasome assembly, probably by interfering with the activation pathways. Recently, Yan et al. reported that omega-3 fatty acids inhibit Results are presented as means ± SEM of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001 by t -test. ND, not determined. diet on infl ammation, but several lines of evidence point in this direction. Numerous studies described a powerful antiinfl ammatory effect for oleate and linoleate. Ex vivo macrophages from rats treated by gavage with oleate or linoleate showed a reduction of cytokine secretion, including IL-1 ␤ , compared with untreated rats ( 52 ), and intracerebroventricular injection of oleate reduced hypothalamic infl ammation in rats ( 53 ). In most cases, intervention studies are performed in animals by changing the diet. Various groups reported an improvement in obesity-associated infl ammation (32)(33)(34) and insulin sensitivity ( 34, 53 ) after a diet rich in UFAs, partially attributing these results to oleate-and linoleate-rich oils. In humans, there is an association between high-SFA and/or low-UFA concentrations and infl ammation ( 54,55 ) or metabolic syndrome ( 54 ). In some studies, these parameters can predict metabolic syndrome development ( 56,57 ). Assays on small groups of people described a decrease of infl ammatory gene expression ( 31 ) and an improvement of insulin sensitivity ( 58 ) after UFA-rich diet. Two large trials have been conducted to assess the benefi t of a high-oleate diet on insulin sensitivity: the KANWU study ( 59 ) and, most recently, the LIPGENE study ( 60 ). In both studies, the oleate diet led to a signifi cant increase in plasma oleate, but only the KANWU study reported an improvement of insulin sensitivity. Contradictory results are common in this type of study in which the design is crucial : choice of subjects (healthy or obese), ethnicity/genetics, initial diet, etc., may all affect the fi nal outcome. In all the studies reporting an effect of UFAs, few mechanisms were proposed. Protection against infl ammation and obesity-associated diseases reported after a high-UFA diet is not yet understood, but NLRP3 infl ammasome inhibition might be a partial explanation.
In conclusion, we demonstrated that SFAs palmitate and stearate trigger IL-1 ␤ secretion in various models of human monocytes and macrophages in a caspase-1/ASC/ NLRP3-dependent pathway. The UFAs oleate and linoleate, which did not activate the NLRP3 infl ammasome, totally prevented the NLRP3 infl ammasome activation induced by SFAs and, with less effi ciency, by a broad range of NLRP3 activators. These results could be correlated with in vivo studies reporting anti-infl ammatory properties of a UFA-rich diet on IL-1 ␤ production. By preventing NLRP3 infl ammasome activation, UFAs might play a protective role in NLRP3-associated diseases, such as type 2 diabetes, atherosclerosis, and gouty arthritis.
Currently, the ATP-activating pathway is commonly described as very simple with few places for UFA interference: ATP binds to P2X7 receptor leading to K+ effl ux and NLRP3 infl ammasome activation ( 50 ). An interesting fi nding reported that crystal activators, such as alum and MSU, trigger NLRP3 infl ammasome activation indirectly through ATP release ( 51 ). If this new mechanism can be extended to all the NLRP3 inducers, one likely explanation could be that UFAs prevent ATP release but have no effect on exogenous ATP.
Although the mechanism is not yet fully elucidated, UFAs could play a protective role in diseases in which NLRP3 is involved, such as type 2 diabetes, atherosclerosis, or gouty arthritis. Unlike treatments with anakinra or other IL-1 ␤ antagonists requiring regular injections, simple dietary interventions could be helpful to obese people in decreasing Il-1 ␤ secretion and infl ammation. Further investigation is required before concluding a protective effect of a high-UFA