TREM-1 governs NLRP3 inflammasome activation of macrophages by firing up glycolysis in acute lung injury

The triggering receptor expressed on myeloid cells-1 (TREM-1) is a pro-inflammatory immune receptor potentiating acute lung injury (ALI). However, the mechanism of TREM-1-triggered inflammation response remains poorly understood. Here, we showed that TREM-1 blocking attenuated NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome activation and glycolysis in LPS-induced ALI mice. Then, we observed that TREM-1 activation enhanced glucose consumption, induced glycolysis, and inhibited oxidative phosphorylation in macrophages. Specifically, inhibition of glycolysis with 2-deoxyglucose diminished NLRP3 inflammasome activation of macrophages triggered by TREM-1. Hypoxia-inducible factor-1α (HIF-1α) is a critical transcriptional regulator of glycolysis. We further found that TREM-1 activation facilitated HIF-1α accumulation and translocation to the nucleus via the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. Inhibiting mTOR or HIF-1α also suppressed TREM-1-induced metabolic reprogramming and NLRP3/caspase-1 activation. Overall, the mTOR/HIF-1α/glycolysis pathway is a novel mechanism underlying TREM-1-governed NLRP3 inflammasome activation. Therapeutic targeting of the mTOR/HIF-1α/glycolysis pathway in TREM-1-activated macrophages could be beneficial for treating or preventing inflammatory diseases, such as ALI.


Introduction
Acute lung injury (ALI), a progressive and devastating clinical condition, is characterized by progressive alveolar-capillary barrier damage, local inflammatory accumulation, denudation of the alveolar epithelium, and hyaline membrane formation [1]. Particularly inflammation plays a crucial role in the pathogenesis of ALI [2]. Macrophages comprise the front line of defense against the pathogen in the lung [3,4]. We have reported that the depletion of macrophages mitigates ALI in mice [3]. The triggering receptor expressed on myeloid cells-1 (TREM-1) is an activating immune receptor, constitutively expressed on monocytes/macrophages and neutrophils [5].
TREM-1, coupled to the adaptor protein 12-kDa DNAX activating protein (DAP12) phosphorylation, activates spleen tyrosine kinase (Syk) and phosphatidylinositol-3 kinase (PI3K), resulting in the production of inflammatory cytokines and chemokines [6]. Our previous study found that the expressions of TREM-1 in lipopolysaccharide (LPS)-induced ALI mice lungs and macrophages are significantly increased, and blocking TREM-1 mitigates LPSinduced ALI [7,8]. NOD-, LRR-and pyrin domain-containing 3 (NLRP3) inflammasome is a cytosolic signaling complex comprising a sensor molecule, the adaptor apoptosis-associated speck-like Ivyspring International Publisher protein containing a CARD (ASC), and the effector protease caspase 1. Once activated, the NLRP3 inflammasome induces pro-caspase-1 self-cleavage and activation, mediating the maturation and secretion of interleukin-1 beta (IL-1β), IL-18, and high-mobility group protein B1 (HMGB1) [9]. These bioactive cytokines play a pivotal role in initiating and amplifying the inflammatory processes during ALI. Other investigators and we have identified that the NLRP3 inflammasome is a critical inflammatory orchestrator during ALI. NLRP3 deficiency alleviates pancreatitis-associated ALI [10]. We have reported intervention factors such as a COX-2/sEH dual inhibitor, vasoactive intestinal peptide (VIP), and epoxyeicosatrienoic acids could attenuate ALI by inhibiting the NLRP3 inflammasome activation in mice [3,11,12]. Recently we have reported blocking TREM-1 attenuates NLRP3 inflammasome activation in LPS-induced ALI [8]. However, the mechanism by which TREM-1 governs NLPR3 inflammasome activation in ALI remains unclear.
Recently glucose metabolic reprogramming has been thought to be a crucial regulator of the NLRP3 inflammasome activation in macrophages [13]. Metabolic reprogramming is first described in cancer cells, also called the Warburg effect, characterized by an increase in aerobic glycolysis and a reduction of oxidative phosphorylation (OXPHOS) [14]. LPSinduced glycolysis stimulates IL-1β expression by hypoxia-inducible factor-1α (HIF-1α), hexokinase-II (HK2), and pyruvate dehydrogenase kinase M2 (PKM2) activation. Those three molecules of glycolysis are directly involved in IL-1β secretion and NLRP3 inflammasome activation [15]. Our study indicates that the blockade of glycolysis partially inhibits the NLRP3 inflammasome activation in LPS-induced ALI [16]. In parallel, the NLRP3 inflammasome activation is correlated with glucose transporter 1 (GLUT1)-dependent glycolysis in postburn [17]. Glycolysis-related increase in reactive oxygen species (ROS) level contributes to the NLRP3 inflammasome activation and IL-1β secretion [18]. So, understanding the role of TREM-1 in regulating glucose metabolic processes is crucial for deciphering how TREM-1 governs NLRP3 inflammasome activation in ALI.
HIF-1α is known to activate transcriptional targets regulating glucose uptake, glycolysis, and flux [19]. Thus, activated HIF-1α increases glucose metabolism through glycolysis but the reduced entry of glucose into the OXPHOS. Such metabolic alteration provides immune cells with increased biomass production, thus fueling inflammation [20]. HIF-1α signaling is essential for macrophagemediated inflammation [21]. HIF-1α binds to a site approximately 300 bp upstream of the transcription start site of IL-1β, inducing Il-1β mRNA expression [22].
Here, we hypothesized that TREM-1 instigated HIF-1α accumulation in PI3K/AKT/mTORdependent manner, resulting in macrophages' glucose metabolic reprogramming, which was critical to NLRP3 inflammasome activation. Our work reveals a novel association between metabolism and inflammation in macrophages.

Lung histology and inflammatory injury score
Six hours after the LPS injection, mice were sacrificed. The left lobe was fixed at 4% neutral buffered formaldehyde solution at 4℃. Multiple sections (4 µm) were sliced for hematoxylin and eosin staining (H&E, Solarbio, China, Beijing). Images were taken with Pannoramic Scan (3Dhistech, Hungary, Budapest). The scoring of histological changes was measured. According to five independent variables, the severity of morphologic criteria was graded from 0 to 4: mixed cell alveolar inflammation, bronchoalveolar hyperplasia, hemorrhage, alveolar lipoproteinosis, and hyaline membranes. The lung injury score was performed by three blinded pathologists.

Isolation and culture of primary mouse peritoneal macrophages
Primary mouse peritoneal macrophages were obtained from C57BL/6J mice. Individual mice were injected with 3 mL 3% sterile thioglycolate (Sigma-Aldrich) intraperitoneally. Three or four days later, macrophages were elicited. Cells were cultured and plated into 6-well plates (2×10 6 cells/well) or 12-well plates (1×10 6 cells/well) in RPMI-1640 (Gibco, Life Technologies, Carlsbad, CA) with 10% heat-inactivated bovine calf serum (BCS, Gibco) at 37 °C for 2 h. After non-adherent cells were washed, the adherent cells were cultured for the following experiments.

Lactate production
Measurement of lactate concentration was utilized with a lactate Assay kit (Sigma-Aldrich) according to the manufacturer's protocol. The main reaction mixture contains a 2 μL lactate probe, 26 μL sample solution, 26 μL lactate assay buffer, and 2 μL lactate mix. The sample was incubated at room temperature for 30 min, and the absorbance was measured at colorimetric (570 nm)/fluorometric (λ ex=535 nm/λ em=587 nm).

Glucose consumption assay
The glucose level in the supernatant was quantified utilizing a high-sensitivity glucose assay kit (Sigma-Aldrich) according to the manufacturer's protocol. The main reaction mixture contains 45 μL glucose assay buffer, 1 μL glucose probe, 2 μL glucose enzyme mix, 2 μL glucose substrate mix, and glucose standard. It was mixed well by pipetting and incubated the reaction for 30 min at 37 °C. The fluorescence intensity was measured at (λ ex =535 nm/λ em =587 nm) using a Varioskan Flash (Thermo Fisher Scientific).

Western blot analysis
Lung tissue homogenate and macrophages were harvested, and proteins were extracted using RIPA buffer (Beyotime) containing protease inhibitors cocktail (Roche, Mannheim, Germany). To concentrate supernatants for western blot, 700 μL 100% methanol and 175 μL trichloromethane were added to 700 μL supernatant and vortexed for 30 s. Supernatants were then centrifuged at 13000 rpm for 5 min at 4°C. The supernatant liquid was removed, and added 700 μL 100% methanol, then centrifuged at 13000 rpm for 5 min at 4°C. Supernatants were discarded. And the remaining pellet was resuspended in 20 μL10% SDS, then added 4 μL 5×SDS-PAGE sample loading buffer (Beyotime) and boiled for 10 min at 95°C. The protein concentrations were measured with Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific, Grand Island, USA).
Equal amounts of protein or all protein from supernatants were subjected to 8%-12% gradient polyacrylic amide gel under reducing conditions. Gels were transferred into polyvinylidene difluoride membranes (Millipore, USA), blocked with 5% fat-free milk or 5% albumin from bovine serum (BSA, Biofroxx, Germany) at room temperature for 1.5 h. The blots were reacted with the primary antibody at 4 °C overnight, followed by horseradish peroxidaseconjugated secondary antibody (1:1000; Cell Signaling Technology, USA), and detection by ChemiDoc XRS (Bio-Rad, USA). The intensities of the bands were quantified using the Image Lab Analyzer software (Bio-Rad). β-actin, α-tubulin, or GAPDH were used as a loading control. The antibodies used in the study are shown in Table 1.

Real-time PCR
Total RNA was isolated from macrophages and lungs using RNAiso (TaKaRa Clontech, Japan). Reverse transcription with approximately 1 μg of total RNA was carried out in a T100 TM Thermal Cycler (Bio-Rad, USA) using PrimeScript TM RT reagent Kit (TaKaRa Clontech). Targeted gene expressions were measured by quantitative real-time PCR analyses using SYBR ® Premix Ex Taq™ II system (TaKaRa Clontech) on a Bio-Rad real-time PCR system (CFX96 Touch™; Bio-Rad, USA). The qPCR program was initiated at 95 °C for 30 s; 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. β-actin was used as an endogenous reference gene. The primers in Table 2 were purchased from Sangon Biotech (Shanghai, China). Gene expression abundance was calculated by the 2 −ΔΔCt method.

Immunofluorescence
To image HIF-1α nuclear translocation and mitochondria morphology, macrophages were stimulated with Mab1187 (10 μg/mL) for 24 h and then washed with PBS three times for 5 min, fixed with 4 % paraformaldehyde for 15 min. The cells were incubated with 0.2% Triton X-100 and blocked with 1% BSA for 30 min before being stained with anti-mouse HIF-1α (1:100, Novus) overnight at 4°C. After washing 3 times, the cells were incubated with Alexa Fluor 488-conjugated anti-mouse IgG antibody overnight at 4°C (ABclonal, China). The nuclei were counterstained with DAPI (Solarbio, China). After that, coverslips were mounted with a drop of ProLong Gold antifade mounting reagent (Solarbio). Images were captured with a fluorescence microscope (Nikon, Tokyo, Japan). HIF-1α-positive regions were determined using ImageJ software.

Evaluation of oxidative stress
Intracellular ROS was measured by a ROS assay (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol. The main reaction mixture contains dichloro-dihydro-fluorescein diacetate and a 10 μM fluorescent indicator of cytosolic ROS. After incubating the reaction for 30 min in the dark, an immunofluorescence photograph was measured using a fluorescence microscope (Nikon, Japan).

Statistical analysis
Data represented in this study were repeated at least three times and expressed as the mean ± SD. Normally distributed data were analyzed using a one-way analysis of variance (ANOVA) for comparisons among multiple groups. Comparisons between the two-group were made with an unpaired t-test. P-value < 0.05 was regarded as statistically significant. Statistical analyses were conducted using SPSS 22.0 (IBM, Chicago, IL) or GraphPad Prism 7.0 (San Diego, CA, USA). N represents experiments performed on individual mice or different macrophages from separate mice. , and Hif-1α mRNA in the lungs was detected by real-time PCR. Data was normalized to housekeeping gene β-actin, n=5-10 mice/group. (J) Glycolysis-associated proteins of HK2, p-mTOR, mTOR, and HIF-1α in the lung lysates were assessed by western blot with α-tubulin as a loading control. (K-N) Quantification of indicated protein levels in (J), n=6-8 mice/group. In all cases, the experiment was repeated twice. Dots represent individual animal values. Statistical analysis was performed using One-way ANOVA adjusted by Tukey's multiple comparison test for Control vs. ALI or ALI vs. LR12+ALI. Error bars indicate mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001. Original western blots represented in graphs are available in Figure 1-source data.

Pharmacologic blockade of TREM-1 attenuated LPS-induced lung injury and glycolysis in mice
It has been reported that blocking TREM-1 partially inhibits NLRP3 inflammasome activation in LPS-induced ALI, and glycolysis is a crucial regulation of NLRP3 inflammasome activation in macrophages. So, we would like to investigate whether blocking TREM-1 glucose metabolism would alter ALI. We first exposed mice to LR12, a TREM-1 decoy receptor, validated in rodents [28]. Histological study showed that LR12-treated mice lungs had less leukocyte infiltration, alveolar congestion, and alveolar barrier thickening than LPS-treated lungs (Figure 1A-B). The levels of IL-1β, IL-1β p17, and nitric oxide synthase (iNOS) in the lungs were significantly decreased in LR12-treated mice ( Figure  1C-F). Then we analyzed changes in gluconic metabolism. We found that the levels of lactate, the end production of aerobic glycolysis, in BALF and serum were similarly decreased in LR12-treated mice (Figure 1G-H). LR12 treatment significantly attenuated the expression of genes encoding enzymes in the glycolytic pathway, including Hk2, fructose-2,6biphosphatase 3 (Pfkfb3), and Hif-1α (Figure 1I). HK2 catalyzes the first step in glucose metabolism. The levels of HK2 in the lungs were decreased in LR12-treated mice (Figure 1J-K). mTOR activation is sufficient to stimulate glycolysis [29]. LR12 treatment significantly reduced the LPS-induced phosphorylated (p)-S2448-mTOR and mTOR levels in the lungs (Figure 1J, L, M). The level of HIF-1α in the lungs was significantly decreased in LR12-treated mice (Figure 1J, N). Together, these data illustrate that TREM-1 inhibition attenuates intrapulmonary inflammation and limits glycolysis.

TREM-1 activation induced glucose metabolic reprogramming in macrophages
To systematically profile metabolic alterations in TREM-1-activated macrophages, we observed lactate production, glucose consumption, and carbohydrate metabolic enzymes. The agonist anti-TREM-1 Ab (Mab1187) has been shown to activate TREM-1 [30]. Notably, TREM-1 activation increased lactate production and glucose consumption (Figure 2A, B). GLUT1 plays an essential role in glucose uptake in macrophages [31]. We showed that TREM-1 activation up-regulated the Glut1 gene expression on macrophages (Figure 2C). In addition, TREM-1 activation was accompanied by increased expression of key glycolytic enzymes, including Hk2, Pfkfb3, Pkm2, lactate dehydrogenase A (Ldha), and glycolysis critical transcription factor HIF-1α in macrophages ( Figure 2C). Protein expression of HK2 and LDHA was also increased in TREM-1-activated macrophages (Figure 2D-F). We further observed that TREM-1 activation was accompanied by a decrease in mitochondrial complex III, IV, and V protein levels (Figure 2G-J). These TREM-1-activated macrophages exhibited a skewed profile, favoring glycolytic factors over oxidative regulators. In addition, coincubation with LR12 partially restored metabolic alteration induced by LPS in macrophages. LPS-mediated increase in lactate production, glucose consumption, and glycolysis genes, such as Glut-1, Ldha, and Hif-1α mRNA expression, was significantly diminished by LR12 (Figure 2K-M). Collectively, these data illustrate that TREM-1-activated macrophages become more glycolytic but less OXPHOS. IL-1β contents in the supernatants were analyzed with ELISA, n=5. n represents experiments performed on different macrophages from separate mice. Bar graphs represent mean ± SD. Student's t-test (two-tailed, unpaired) was used to compare Control and Mab1187: * P < 0.05, ** P < 0.01, and *** P < 0.001.

Functional HIF-1α expression was induced by TREM-1 activation in macrophages
We next investigated how TREM-1 activation induced glycolysis in macrophages. HIF-1α mediates metabolic reprogramming towards a glycolytic phenotype by inducing the expression of glycolytic enzymes, such as Glut-1, HK2, PKM2, and LDH [34]. We found that TREM-1-activated-macrophages expressed significantly higher levels of HIF-1α mRNA and protein, even under normoxic conditions ( Figure  5A-C). Furthermore, as a functional consequence, HIF-1α accumulation and translocation to the nucleus were also significantly increased in the TREM-1 activation group through immunofluorescence (Figure 5D-E). Macrophages treated with PX-478, a HIF-1α inhibitor, significantly inhibited HIF-1α accumulation and translocation to the nucleus induced by TREM-1 activation (Supplementary Figure 3A). PX-478 treatment displayed a decreased protein level of HK2, a HIF-1α target gene, in response to TREM-1 activation (Figures 5F-G). These results indicate that TREM-1 activation promotes HIF-1α stabilization in macrophages even under normoxic conditions.

Discussion
In this study, we found that TREM-1 activation induced a glucose metabolic reprogramming of macrophages via mTOR/HIF-1α signaling, which triggered NLRP3 inflammasome activation in ALI (Figure 9). Our previous study found blocking TREM-1 partially inhibits NLRP3 inflammasome activation in ALI. Then in this study, we observed that blocking TREM-1 also limited glycolysis in ALI mice.
The crosstalk between TREM-1 and NLRP3 inflammasome has emerged as a novel mechanism of the inflammatory cascade in ALI. We have reported that TREM-1 blockade with LR12 inhibits the NLRP3 inflammasome activation in ALI [8]. Others have shown that TREM-1 inhibition with synthetic peptide LP17 ameliorates neuroinflammatory injury and chronic obstructive pulmonary disease (COPD) via NLRP3 inflammasome-mediated pyroptosis [37,38]. TREM-1 blockade with LP17 restrains NLRP3/ caspase-1 activation through SYK in microglia [39]. Recent studies have shown that TREM-1 serves as a receptor for extracellular cold-inducible RNA-binding protein (eCIRP) to induce inflammation in ALI [40]. Previous studies have been performed with TREM-1 inhibitory peptides, which was an indirect effect.
Here, we first demonstrate that TREM-1 signaling using an agonist anti-TREM-1 Ab (Mab1187) promoted the priming and activation of NLRP3 inflammasome in macrophages. Our recent studies identified NLRP3 inflammasome as a new trigger of TREM-1 signaling. HMGB1 and IL-18 released by NLRP3 inflammasome triggered the TREM-1amplified response [41]. Those findings suggest that TREM-1 and NLRP3 inflammasome forms a positive feedback loop, promoting pulmonary inflammation. . n=3 biological replicates. Statistical analysis was performed using One-way ANOVA adjusted by Tukey's multiple comparison test. Data were expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.  . (A-B) Western blot and quantification of p-DRP1 ser616 and DRP1 in control or 24 h TREM-1-activated macrophages. n=3 biological replicates. Statistical analysis was performed using Student's t-test (two-tailed, unpaired). Macrophages were premixed with PBS control or Mdivi-1 (100 nM) before incubating with plate-bound agonistic anti-TREM-1 mAb for 24 h. (C) Protein levels of NLRP3, pro-IL-1β, IL-1β p17, and caspase-1 p10 in macrophages were detected by western blot. (D-G) Quantification of NLRP3, pro-IL-1β, IL-1β p17, and caspase-1 p10 (C), n=3 biological replicates. Statistical analysis was performed using One-way ANOVA. Data were expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001. Our study clarified the induction mechanism of glucose metabolism reprogramming during ALI. We have reported that glycolysis is a deteriorative factor in ALI [16]. While its induction mechanism has not been thoroughly elucidated. Herein, our findings identify TREM-1 as a glycolytic stimulus, as evidenced by accentuated glucose consumption and lactate secretion and up-regulation of glycolytic metabolic enzymes in macrophages. Blocking TREM-1 attenuated intrapulmonary inflammation and limited glycolysis. Moreover, TREM-1 was found to promote mitochondrial fission in macrophages by facilitating DRP1 s616 phosphorylation. Mitochondrial dynamic controls macrophages' fate through metabolic programming [42]. Fission in macrophages leads to cristae expansion, reducing electron transport chain (ETC) efficiency and decreasing OXPHOS [43]. These results are in line with an observation from IL-34, where IL-34 reprograms naïve myeloid cells into glycolytic macrophages [44]. Also reported, most microbial stimuli increase glycolysis, but only stimulating the TLR4 with LPS leads to an increase in glycolysis. Instead, stimulation of TLR2 increases oxygen consumption and mitochondrial enzyme activity in monocytes [45]. Furthermore, we found that TREM-1 instigates metabolic reprogramming in a PI3K/AKT/HIF-1α-dependent manner. Interestingly, ROS scavenger did not influence TREM-1 activated-HIF-1α. This observation is in line with the observation that mitochondrial, cytosolic, or lipid ROS were unnecessary for HIF-1α stability and transcription [46].
Glucose metabolites are thought to regulate the activation of the NLRP3 inflammasome [13]. Here, we found that TREM-1 triggers NLRP3/caspase-1 activation and promotes IL-1β secretion. Using glycolysis inhibitor 2-DG reduced TREM-1 activation induced-NLRP3 inflammasomes. Glycolysis not only provides energy but also intermediates to work as a signal molecule activating NLRP3 inflammasome, e.g., Up-regulation of HK1-dependent glycolysis by mTOR regulates NLRP3 inflammasome activation [15]. PKM2-dependent glycolysis promotes NLRP3 inflammasome activation by modulating the eukaryotic translation initiation factor 2 alpha kinase 2 phosphorylation in macrophages [47]. Lactate is essential for NLRP3 inflammasome activation [48]. Our results showed that inhibition of glycolysis with 2-DG suppresses TREM-1 protein in mice with ALI induced by LPS. Our previous studies showed that inflammatory response precedes enhanced glycolysis during the development of ALI, and inflammation could induce glycolysis [16]. Glycolysis and its intermediate metabolites are involved in inflammation as signaling molecules. In addition, Glycolysis, potentially through reactive aldehydes and a redox-dependent mechanism, exerts positive feedback on the inflammatory transcription factors [49]. Intermediate metabolites of glycolysis can promote the expression of TREM-1 ligands and inducers. For example, PKM2, a key enzyme in glycolysis, regulates the Warburg effect and promotes HMGB1 release [47,50]. HMGB1 has been suggested as a TREM-1 ligand [51]. PKM2 promotes cyclooxygenase (COX)-2 [52] and HIF-1α-dependent transcriptional up-regulation of COX-2, which regulates the expression of TREM-1 [53]. This result suggests that inhibiting glycolysis reduced the expression of the TREM-1 ligand or inducer by inhibiting glycolysis. HIF-1α, a key transcription factor in glycolysis, mediates NLRP3 inflammasome activation in synovial fibrosis [54]. PI3K inhibitor attenuates NLRP3 inflammasome activation in neural stem cells [55]. Our results also found inhibition of the mTOR signal or HIF-1α reduced TREM-1-induced NLRP3 inflammasome activation. Collectively, the findings indicate that TREM-1 triggers NLRP3 inflammasome in an mTOR/HIF-1α/glycolysisdependent manner.
Our preclinical data are promising in the therapeutic potential of TREM-1, while its downstream metabolic intermediates remain to be elucidated. Further studies are needed to understand how glycolysis participates in TREM-1-induced NLRP3 inflammasome. N-acetylglucosamine reportedly promotes untethering of HK2 from the mitochondria, which is sufficient to drive NLRP3 inflammasome activation [56]. The mechanisms of mTOR, HK2, and HIF-1α involved in TREM-1triggered NLRP3 inflammasome activation require further exploration.
In conclusion, our study reveals a crucial role of TREM-1 in controlling glucose metabolism via the HIF-1α pathway. This glucose metabolic reprogramming by TREM-1 is vital to the NLRP3 inflammasome activation. The mTOR/HIF-1α/glycolysis pathway in macrophages may thus be a novel strategy for the treatment of TREM-1-and NLRP3 inflammasomeassociated inflammatory diseases.