ABSTRACT Acute lung injury (ALI) as well as its more severe form, acute respiratory distress syndrome (ARDS), frequently leads to an uncontrolled inflammatory response. N6-methyladenosine

(m6A) modification was associated with the progression of several inflammatory diseases. However, the role of methyltransferase-like 14 (METTL14)-mediated m6A methylation in ALI/ARDS remains

unclear. Here, we reported an increase in overall expression levels of m6A and METTL14 in circulating monocyte-derived macrophages recruited to the lung following ALI, which is correlated

with the severity of lung injury. We further demonstrated the critical function of METTL14 in activating NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome in vitro and

knockdown diminished LPS-induced ALI in mice by downregulating NLRP3 expression. In summation, our study revealed that the molecular mechanism underlying the pathogenesis of ALI/ARDS

involves METTL14-mediated activation of NLRP3 inflammasome in an IGF2BP2 dependent manner, thereby demonstrating the potential of METTL14 and IGF2BP2 as promising biomarkers and therapeutic

targets for ALI/ARDS treatment. SIMILAR CONTENT BEING VIEWED BY OTHERS NUCLEAR SPHK2/S1P INDUCES OXIDATIVE STRESS AND NLRP3 INFLAMMASOME ACTIVATION VIA PROMOTING P53 ACETYLATION IN

LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG INJURY Article Open access 18 January 2023 DECODING THE MULTIPLE FUNCTIONS OF ZBP1 IN THE MECHANISM OF SEPSIS-INDUCED ACUTE LUNG INJURY Article Open

access 21 October 2024 TETRAMETHYLPYRAZINE AMELIORATES LPS-INDUCED ACUTE LUNG INJURY VIA THE MIR-369-3P/DSTN AXIS Article Open access 28 August 2024 INTRODUCTION Acute lung injury (ALI) and

its more severe form, acute respiratory distress syndrome (ARDS) are common, life-threatening critical illnesses that lead to significant morbidity and mortality [1]. Despite prominent

breakthroughs in the pathophysiology of ALI/ARDS, the hospital mortality rate of these disorders remains high (46.1%), and effective pharmacological treatments are still lacking [2].

ALI/ARDS is characterized by dysregulated lung parenchymal inflammation, leading to diffuse alveolar damage and edema, ultimately contributing to acute hypoxemic respiratory failure [3].

Uncontrolled local or systemic inflammation is believed to be the predominant cause of ALI/ARDS [4]. Activated macrophages, especially recruited circulating monocyte-derived macrophages

further release pro-inflammatory cytokines, which give rise to an inflammation cascade [5]. The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is excessively

activated in macrophages during ALI/ARDS progression [6]. The NLRP3 inflammasome, which consists of a sensor (NLRP3), an adaptor apoptosis-associated speck-like protein containing a

caspase-recruitment domain (ASC), and an effector caspase (caspase-1), is involved in the production of pro-inflammatory cytokines, interleukin (IL)-1β and IL-18 [7]. NLRP3 inflammasome

activation reportedly involves two steps: priming and activation [8]. The priming step of NLRP3 inflammasome activation is regulated via transcriptional and post-translational mechanisms

[9]. NF-κB signaling induces the transcriptional activation of NLRP3 priming by upregulating the gene expression of NLRP3 inflammasome components [10, 11]. Post-translational modifications

(PTMs) of NLRP3, such as ubiquitination, phosphorylation, and SUMOylation, may stabilize NLRP3 in an auto-suppressed inactive state [12]. The activation step is induced by various

pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), including extracellular ATP, pore-forming toxins, RNA viruses, and particulate matter [13].

However, post-transcriptional regulation of NLRP3 inflammasome activation in ALI/ARDS remains unclear. N6-methyladenosine (m6A) modification, the most abundant modification of messenger RNA

(mRNA), may reversibly regulate target genes at the post-transcriptional level, thereby affecting almost all crucial biological processes [14]. This dynamic and reversible process is

primarily regulated by the m6A methyltransferase complex, which contains methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms tumor suppressor-1-associated protein

(WTAP), and demethylases, including fat mass and obesity-related protein (FTO) and ALKB homolog 5 protein (ALKBH5) [15]. Meanwhile, RNA-binding proteins that identify and bind to m6A sites,

such as the YT521-B homology domain (YTHD) family, and the insulin-like growth factor 2 mRNA-binding protein (IGF2BP) family, serve as m6A readers and direct the fate of target RNAs by

influencing alternative pre-mRNA splicing, RNA stability, and translation efficiency [16]. m6A facilitates the progression of several inflammatory diseases, such as non-alcoholic fatty liver

disease, autoimmune diseases, and infections [17,18,19,20]. Evidently, global m6A levels are significantly increased in alveolar epithelial cells, mediated by the upregulation of METTL3,

which is closely associated with ALI [21]. Nonetheless, the effects of METTL14-regulated m6A methylation in ALI/ARDS remain unclear and the precise molecular targets of METTL14 in ALI/ARDS

remain to be elucidated. Therefore, we sought to determine the functional role of METTL14 and its target in ALI/ARDS. Herein, we first elucidated that RNA m6A modification in macrophages is

involved in the progression of ALI/ARDS, and then verified the role of METTL14 in this process. Further mechanistic studies revealed that NLRP3 is the methylated target of METTL14 and that

IGF2BP2 stabilizes NLRP3 mRNA during NLRP3 inflammasome activation in ALI/ARDS. These results indicated that METTL4 together with IGF2BP2 may be promising therapeutic targets in ALI/ARDS.

RESULTS GLOBAL M6A LEVELS AND METTL14 EXPRESSION ARE INCREASED IN ALI MICE To confirm the role of m6A modification in ALI/ARDS, we evaluated the global m6A levels in the lung tissues of

control and ALI mice. Both dot blot assay and colorimetric quantification showed that global m6A levels in the total RNA were significantly increased in ALI lungs compared to the control

group (Fig. 1A–C). We then detected the expression of m6A methyltransferases (METTL3, METTL14, METTL16, and WTAP) and demethylases (FTO and ALKBH5) in lung tissues. The expression of METTL14

mRNA and METTL14 protein was markedly upregulated in ALI mice, whereas no significant differences were found in the expression of other regulators (Fig. 1D–F). These results indicated that

METTL14-mediated m6A methylation may play a functional role in ALI/ARDS. Subsequently, we employed immunofluorescence staining to identify the specific cell types involved in ALI/ARDS that

express METTL14. Our findings revealed co-localization of METTL14 not only with CD68-labeled macrophages but also with CK7-labeled pulmonary epithelial cells. Interestingly, compared with

sham group, ALI lungs exhibited an increased number of METTL14-expressed CD68+ macrophages, while the number of METTL14-expressed CK7+ epithelial cells did not show a significant change

(Figs. 1G, H and S1A, B). To clarify the origins of elevated METTL14+ macrophages, quantitative analysis unveiled that METTL14+/ F4/80+, rather than METTL14+/Siglec-F+ (a marker for resident

alveolar macrophage) cells increased in ALI mice compared with the corresponding sham mice (Fig. 1I–L). Collectively, these findings indicated that the expression level of METTL14 is

elevated in LPS-induced ALI model, particularly in recruited circulating monocyte-derived macrophages within the lung. GLOBAL M6A LEVELS AND METTL14 EXPRESSION ARE INCREASED IN LPS-ACTIVATED

MACROPHAGES The NLRP3 inflammasome is a crucial factor in triggering the activation of macrophages during this pathogenesis [22]. We subsequently assessed the expression level of METTL14

within a NLRP3 inflammasome activation model in RAW264.7 macrophages. As expected, treatment of RAW264.7 macrophages with LPS and nigericin significantly increased the release of

NLRP3-inflammasome-dependent cytokines, including IL-1β p17, Caspase-1 p20, IL-1β and IL-18 (Fig. 2A, E, F). The mRNA and protein levels of METTL14 and NLRP3 in RAW264.7 macrophages were

observably enhanced following stimulation with LPS whether combined with nigericin or not, indicating METTL14 may participate in the priming step (Fig. 2A–D). Both dot blot assay and

colorimetric quantification showed that global m6A levels of total RNA in activated macrophages were notably elevated (Fig. 2G–I). Our findings pointed towards a possible role for

METTL14-mediated m6A methylation in the process of NLRP3 inflammasome activation. KNOCKING DOWN METTL14 INHIBITS THE ACTIVATION OF NLRP3 INFLAMMASOME AND ALLEVIATES LUNG INJURY IN VITRO AND

IN VIVO To determine the function of METTL14, we knocked down METTL14 expression in RAW264.7 macrophages using small interfering RNA (siRNA). Western blotting, real-time PCR, and

colorimetric m6A quantification were used to verify the knockdown effect (Fig. 3A–D). Considering si-METTL14 #3 exhibited the superior knockdown efficiency, it was selected for further

experiments. Compared with the negative control (si-NC) cells, knockdown of METTL14 exhibited a significant decrease in NLRP3 protein expression, as well as a reduced release of IL-1β and

IL-18 cytokines in LPS-stimulated macrophages (Fig. 3E–H). Interestingly, METTL14 knockdown only downregulated the mRNA expression of NLRP3, but not that of IL-1b or IL-18 in LPS-treated

cells (Fig. 3I–K). These results suggested that METTL14 may mediate NLRP3 inflammasome activation via regulating NLRP3. We next employed METTL14 siRNA to determine the in vivo function of

METTL14 in ALI. Total m6A levels in ALI mice were reduced after METTL14 was knocked down (Fig. 3L), suggesting that m6A modification occurring in ALI/ARDS was mainly mediated by METTL14.

Both METTL14 siRNA and MCC950 (NLRP3 inhibitor) administration decreased the lung wet/dry ratio in ALI mice, indicating an alleviation of pulmonary edema associated with ALI/ARDS. (Fig. 3M).

Compared with ALI group, the total protein concentrations in BALF and myeloperoxidase (MPO) activity in lung tissues of si-METTL14 + ALI and MCC950 + ALI group were notably lower (Fig. 3N,

O). Similarly, H&E staining showed relatively intact alveolar structure and less inflammatory cell infiltration in si-METTL14 + ALI and MCC950 + ALI group than those in the ALI group

(Fig. 3P, Q). Consistent with the in vitro results, we found that METTL14 knockdown inhibited the activation of NLRP3 inflammasome in ALI mice via regulating the mRNA levels of NLRP3, rather

than IL-1b and IL-18 (Figs. 3R–T and S2A–D). Collectively, these results suggested that METTL14 knockdown may inhibit NLRP3 inflammasome activation and alleviate lung injury in vitro and in

vivo, confirming that METTL14 plays a vital role in NLRP3 inflammasome activation in ALI/ARDS. ELEVATED METTL14 PROMOTES THE ACTIVATION OF NLRP3 INFLAMMASOME AND AGGRAVATES LUNG INJURY IN

VITRO AND IN VIVO To further elucidate the function of METTL14 in ALI, we performed gain-of-function assay by overexpressing METTL14 in RAW264.7 macrophages (Fig. 4A–C). METTL14

overexpression in RAW264.7 macrophages increased m6A levels (Fig. 4D) and activated NLRP3 inflammasome in macrophages by upregulating the mRNA levels of NLRP3, rather than IL-1b and IL-18

(Fig. 4E–K). We subsequently explored the in vivo function of METTL14 in ALI using AAV9 that expressed full-length METTL14. AAV-GFP was used as a control. As shown in Fig. 4L, a marked

increase in the level of m6A modification was detected in lung tissue of ALI mice. AVV-METTL14 aggravated pulmonary edema of ALI mice, as revealed by lung wet/dry ratio (Fig. 4M). METTL14

overexpression also increased the total protein concentrations in BALF and MPO activity (Fig. 4N, O). Likewise, H&E staining showed thicker alveolar walls and more inflammatory

infiltration in AVV-METTL14 + ALI group than those in the ALI group (Fig. 4P, Q). Indeed, METTL14 activated NLRP3 inflammasome via upregulating the mRNA levels of NLRP3 (Fig. 4R–V). Taken

together, these data supported that the elevation of METTL14 contributes to the activation of the NLRP3 inflammasome and the exacerbation of lung injury in vitro and in vivo. NLRP3 IS THE

DIRECT TARGET OF METTL14-MEDIATED M6A MODIFICATION NLRP3 is present in low concentrations under resting conditions, which is insufficient to activate the inflammasome [11]. Based on previous

results showing that METTL14 regulated the mRNA expression of NLRP3 both in vivo and in vitro, we surmised that NLRP3 may be a direct target of METTL14. To validate the role of m6A

methylation modulated by METTL14 in NLRP3 transcript, we analyzed potential m6A targeting motifs using SRAMP (http://www.cuilab.cn/sramp). A total of 24 RRACH m6A-binding motifs were

identified in NLRP3 mRNA sequence (Fig. 5A and Supplemental Table 3). The m6A RNA immunoprecipitation (MeRIP) assays confirmed that NLRP3 mRNA m6A modification was enhanced in ALI mice and

LPS-treated macrophages (Fig. 5B, D), but significantly decreased after METTL14 knockdown (Fig. 5C, E). Next, we used RNA pull-down and RNA immunoprecipitation (RIP) assays to determine

whether there is a direct interaction between NLRP3 mRNA and METTL14. RNA pull-down assays showed that METTL14 interacts with the NLRP3 transcript, and that this interaction was enhanced in

ALI mice (Fig. 5F). RIP analysis with the METTL14 antibody further confirmed the interaction between METTL14 and NLRP3 mRNA both in vivo and in vitro (Fig. 5G, H). Moreover, rescue assays

were performed by using MCC950, an NLRP3 inhibitor, in AAV-METTL14 mice. Our data revealed that the extent of lung injury in AAV-METTL14 ALI mice was restored by MCC950 treatment, as

revealed by lung wet/dry ratio, BALF protein content, MPO activity and histological injury score (Fig. 5I–M). The over-release of IL-1β and IL-18 in lung tissues and serum in AAV-METTL14 ALI

mice was reversed by MCC950 treatment (Fig. 5N–Q). These findings indicated that NLRP3 is a direct and functional target of METTL14-mediated m6A modification during NLRP3 inflammasome

activation in ALI/ARDS. IGF2BP2 IS UPREGULATED AND ENHANCES THE STABILITY OF NLRP3 MRNA IN ALI Considering that METTL14 induces NLRP3 mRNA m6A methylation and that the loss of m6A in NLRP3

mRNA mediated by METTL14 knockdown leads to a decrease in NLRP3 mRNA and protein expression in ALI mice, we sought to determine whether METTL14-mediated m6A modification affects the NLRP3

mRNA stability. We treated RAW264.7 macrophages with the transcription inhibitor actinomycin D (ActD) and found that NLRP3 decay in si-METTL14-treated macrophages was faster than that in

corresponding controls when stimulated with LPS (Fig. 6A), suggesting that METTL14 regulates NLRP3 expression via an m6A-dependent mRNA decay mechanism. Therefore, we identified m6A readers

that may participate in the regulation of NLRP3 mRNA stability. The IGF2BP family regulates the stability of methylated mRNA by acting as m6A readers [23]. First, we detected the protein

expression of IGF2BP1, IGF2BP2, and IGF2BP3 using western blotting. We found that protein expression of IGF2BP2 was distinctly upregulated in ALI mice (Fig. 6B, C), which was consistent with

its mRNA expression (Fig. 6D). Similarly, the protein and mRNA expression levels of IGF2BP2 were notably augmented in LPS-treated RAW264.7 macrophages (Fig. 6E–G). To further validate the

direct interaction between NLRP3 mRNA and IGF2BP2, we performed an in vivo RNA precipitation assay using a biotinylated NLRP3 probe. RNA pull-down assay detected that specific binding of

IGF2BP2 was enhanced in ALI mice (Fig. 6H). RIP analysis with the IGF2BP2 antibody further confirmed that their interaction was facilitated in vivo and in vitro during ALI (Fig. 6I, J).

These results implied that the stability of NLRP3 mRNA might be regulated by IGF2BP2 via METTL14-mediated m6A modification. IGF2BP2 KNOCKDOWN DECREASES NLRP3 MRNA STABILITY AND INHIBITS

NLRP3 INFLAMMASOME ACTIVATION IN LPS-ACTIVATED MACROPHAGES To examine whether IGF2BP2 regulates NLRP3 expression, we used siRNA to knockdown IGF2BP2 in RAW264.7 macrophages (Fig. 7A–C), and

si-IGF2BP2 #2 with the best knockdown effect was selected for further experiments. As shown, IGF2BP2 knockdown significantly downregulated the mRNA expression of NLRP3 (Fig. 7D), but not

IL-1b (Fig. 7E) or IL-18 (Fig. 7F), in LPS-treated RAW264.7 macrophages. To further determine the mechanism underlying IGF2BP2-induced regulation of NLRP3, we examined the effect of IGF2BP2

knockdown on the lifetime of NLRP3 mRNA. We found that the stability of NLRP3 mRNA in LPS-treated RAW264.7 macrophages was reduced by IGF2BP2 knockdown (Fig. 7G). As expected, IGF2BP2

knockdown inhibited NLRP3 expression (Fig. 7H, I) and the release of IL-1β and IL-18 (Fig. 7J, K) in LPS-treated RAW264.7 macrophages. We proceeded to silence IGF2BP2 in

METTL14-overexpressing cells. Our data showed that IGF2BP2 knockdown reduced NLRP3 mRNA lifespan (Fig. 7L) and restored the over-release of IL-1β and IL-18 (Fig. 7M, N) in

METTL14-overexpressing macrophages. These results confirmed that IGF2BP2 participates in METTL14-mediated NLRP3 inflammasome activation by enhancing the stability of NLRP3 mRNA in

macrophages. KNOCKING DOWN IGF2BP2 INHIBITS THE ACTIVATION OF NLRP3 INFLAMMASOME AND ALLEVIATES LUNG INJURY IN ALI MICE We further determined the therapeutic potential of IGF2BP2 against

mouse ALI models by applying siRNA to knock down IGF2BP2 in vivo. Compared to mice treated with control siRNA, the si-IGF2BP2 group showed significantly alleviated lung wet/dry ratio in ALI

mice (Fig. 8A). IGF2BP2 inhibition also decreased the total protein levels in BALF and MPO activity in ALI lung (Fig. 8B, C). Similar effects of IGF2BP2 knockdown on alleviating lung injury

in ALI mice were revealed H&E staining (Fig. 8D, E). Disruption of IGF2BP2 downregulated the mRNA expression of NLRP3 in the lung tissues of ALI mice (Fig. 8F). The dramatic increase in

the IL-1β and IL-18 levels were efficiently diminished in ALI mice after treated with si-IGF2BP2 (Fig. 8G–J). We further performed IGF2BP2 inhibition in AAV-METTL14 mice. The deterioration

of lung wet/dry ratio, BALF protein content, MPO activity in AAV-METTL14 ALI mice were restored by IGF2BP2 knockdown (Fig. 8K-M). IGF2BP2 inhibition also reduced the upregulation of IL-1β

and IL-18 levels in lung tissues and serum (Fig. 8N–Q) and NLRP3 mRNA (Fig. 8R) in AAV-METTL14 ALI mice. These results manifested that IGF2BP2 knockdown may relieve ALI via inhibiting the

NLRP3 inflammasome activation in vivo. DISCUSSION In this study, we discovered that the contents of m6A and METTL14 in lung tissues of ALI mice subjected to LPS were enhanced.

METTL14-mediated NLRP3 mRNA m6A modification increases NLRP3 mRNA stability in injured lungs in an IGF2BP2-dependent manner. Thus, knocking down METTL14 or IGF2BP2 may play a protective role

in ALI by inhibiting NLRP3 inflammasome activation. This finding may provide new pathophysiological insights into potential therapeutic strategies for ALI/ARDS. N6-Methyladenosine (m6A) is

the most abundant epigenetic mRNA modification and exerts different biological effects in various diseases via post-transcriptional regulation [24,25,26]. Emerging evidence indicates that

m6A may play an indispensable role in some inflammatory diseases [27]. RNA m6A modification is mediated by m6A writers (methyltransferases), erasers (demethylases), and readers. METTL14, a

key component of the m6A methyltransferase complex, stabilizes the structure of METTL3 and enhances its enzymatic activity by binding to RNA, which ultimately increases m6A level [28]. Our

study showed that m6A modification and the m6A methyltransferase METTL14 were increased in ALI mice. Further analysis confirmed METTL14 is mainly elevated in recruited circulating

monocyte-derived macrophages of ALI mice. Interestingly, some studies have shown that neutrophil extracellular traps (NETs) induced ferroptosis in alveolar epithelial cells of cecal ligation

and puncture (CLP)-mouse model by activating METTL3, while a few studies showed METTL3-mediated m6A modification alleviated ALI via inhibiting endothelial injury, indicating that m6A may

exert different effects on ALI/ARDS owing to cell types and challenges [29,30,31]. Alveolar macrophages (AMs) consist of two subpopulations, including resident AMs and recruited AMs [32].

The resident AMs serve as an immunosuppressive subpopulation and mainly present the M2 phenotype, whereas the recruited AMs, which are derived from circulating monocytes, prefer to

differentiate into pro-inflammatory M1 phenotype [33, 34]. Consistent with the enrichment of METTL14 in recruited macrophages, we found the m6A levels and METTL14 expression were also

increased in a RAW264.7 macrophage NLRP3 inflammasome activation model. This finding was in line with recent studies showing that METTL14 activated M1 polarization of macrophages in ischemic

stroke and coronary heart disease, indicating that METTL14 may play a vital role in the functional regulation of macrophages [35, 36]. Uncontrolled inflammatory responses mediated by

pulmonary macrophages are indeed crucial in the pathogenesis of ALI/ARDS [37]. In the present study, we found that METTL14 knockdown alleviated lung injury via inhibiting NLRP3 inflammasome

activation in macrophages, consistent with the result in sepsis-associated myocardial dysfunction [38]. The NLRP3 inflammasome, which acts as the core of the inflammatory response, mediates

caspase-1 activation and the secretion of proinflammatory cytokines, IL-1β/IL-18 [39]. Enhanced activation of the NLRP3 inflammasome in alveolar macrophage is involved in the pathogenesis of

ALI/ARDS caused by various pathogenic factors [40, 41]. Inhibition NLRP3 inflammasome using the specific inhibitor MCC950 has achieved satisfactory therapeutic results not only in ALI model

but also other inflammatory conditions including autoimmune diseases [42]. However, the liver toxicity of MCC950 was found in a phase II clinical trial for rheumatoid arthritis, which casts

a shadow over the future clinical application of MCC950 [43]. In our study, we found that the protective effects of METTL14 knockdown were similar to MCC950 in ALI. Therefore, it is

promising to develop a specific inhibitor of METTL14 for treatment on ALI/ARDS and other inflammatory diseases. Although some studies have revealed an association between METTL14 and NLRP3

inflammasome activation [44, 45], whether METTL14 plays a direct role in regulating NLRP3 expression remains unclear. NLRP3 in low concentrations is inadequate for initiating inflammasome

activation under resting conditions [9]. Our results revealed that METTL14 knockdown markedly downregulated the mRNA expression of NLRP3, but not that of IL-1b or IL-18, both in vivo and in

vitro. Therefore, we suspected that NLRP3 mRNA may be the m6A methylation target of METTL14 in ALI/ARDS. Hence, we analyzed potential m6A targeting motifs in SRAMP and identified 24 RRACH

m6A-binding motifs in NLRP3 mRNA. We further confirmed that the loss of METTL14 abolished the increase in m6A methylation levels of NLRP3 mRNA in ALI mice and RAW264.7 macrophages treated

with LPS. RNA pull-down and RIP assays confirmed that METTL14 directly interacted with NLRP3 mRNA, and that such binding was enhanced in ALI mice. These results indicated that NLRP3 mRNA is

a direct m6A methylation target of METTL14 during NLRP3 inflammasome activation in ALI/ARDS. The biological functions of m6A modifications rely on m6A readers, which regulate RNA metabolism,

including translation, splicing, export, and degradation [16]. Elevated m6A modification mediated by METTL14 increases NLRP3 mRNA and protein expression in ALI mice. Furthermore, an ActD

RNA stability assay showed that the half-life of NLRP3 transcripts had decreased following METTL14 knockdown, indicating that NLRP3 expression was modulated via an m6A-dependent mRNA decay

mechanism. The m6A readers, IGF2BP1/2/3, are involved in regulating the stability of methylated mRNA [46]. Based on our data indicating that only IGF2BP2 was upregulated in ALI mice and

LPS-treated RAW264.7 macrophages, we hypothesized that IGF2BP2 may act as the potential binding protein of NLRP3 mRNA via an m6A-dependent mRNA decay mechanism. Indeed, RNA pull-down and RIP

assays confirmed that IGF2BP2 directly binds to NLRP3 transcripts. Moreover, our findings suggested that IGF2BP2 knockdown may decrease the NLRP3 mRNA stability and inhibit NLRP3

inflammasome activation in ALI mice and LPS-treated RAW264.7 macrophages, thereby alleviating lung injury. Collectively, these results suggest that IGF2BP2 specifically binds to the NLRP3

transcripts and enhances NLRP3 mRNA stability in an m6A-dependent manner during ALI/ARDS. This study has some limitations. We investigated the role of METTL14/IGF2BP2 in NLRP3 inflammasome

activation in ALI mice and RAW264.7 macrophages. However, the role of METTL14/IGF2BP2 in clinical patients of ARDS remains to be elucidated. In addition, although we verified that m6A

modification of NLRP3 mRNA was mediated by METTL14, the specific motif of NLRP3 transcripts methylated by METTL14 has not yet been elucidated and may have to be confirmed in future research.

Third, although we found that METTL14 and IGF2BP2 were upregulated in ALI mice and RAW264.7 macrophages, upstream mechanisms underlying this process have not yet been explored and require

further investigation via a follow-up study. Overall, our study provides robust in vitro and in vivo evidence supporting the critical roles of METTL14/IGF2BP2 in NLRP3 inflammasome

activation during ALI/ARDS. Mechanistically, METTL14-catalyzed NLRP3 mRNA m6A methylation enhances the stability of NLRP3 mRNA in an IGF2BP2-dependent manner in ALI/ARDS. Our findings

indicate that METTL14/IGF2BP2 shows potential as therapeutic targets in the treatment of ALI/ARDS. MATERIALS AND METHODS ANIMALS Male specific-pathogen-free C57/BL6 mice (8–10 weeks old)

were obtained from the Guangdong Medical Laboratory Animal Center (Guangzhou, China). All mice were housed under controlled, pathogen-free conditions at the Laboratory Animal Center of Sun

Yat-sen University Cancer Center. All animals were housed in separate cages in a temperature- (24 ± 1 °C) and humidity-controlled (50–60%) room under a 12/12-h light/dark cycle. All

experiments were conducted in accordance with the guidelines defined by the Sun Yat-sen University Cancer Center. The study was approved by the Animal Care and Ethics Committee of Sun

Yat-sen University Cancer Center (Permit Number: 2021-000043). ANIMAL MODELS AND TREATMENTS A mouse LPS-induced ALI model was established as previously described [47]. Briefly, mice from the

ALI groups were treated with a single intraperitoneal dose of 15 mg/kg LPS obtained from Escherichia coli 055: B5 (Sigma-Aldrich, St. Louis, MO, USA) in saline, whereas mice injected with

the same volume of saline served as controls. After 24 h, the mice were killed and the lung lobes were harvested for further analysis. This in vivo study was performed via six series of

experiments. Mice in the first series were randomly divided into control and ALI groups. Mice in the second series were randomly assigned to receive the following treatments: (1) control +

si-NC, (2) control + si-METTL14, (3) control + MCC950, (4) ALI + si-NC, (5) ALI + si-METTL14 and (6) ALI + MCC950 (an NLRP3 inflammasome inhibitor, 50 mg/kg, i.p., Selleck, Shanghai, China).

Mice in the third series were grouped as follows: (1) control + si-NC, (2) control + si-IGF2BP2, (3) ALI + si-NC, or (4) ALI + si-IGF2BP2. Each group received a dose of 20 nmol siRNA

(either si-NC, si-METTL14 or si-IGF2BP2) in 200 μl of saline via the tail vein 2 d before being challenged with LPS or saline. Mice in the fourth series were grouped as follows: (1) control

+ AAV-GFP, (2) control + AAV-METTL14, (3) ALI + AAV-GFP, or (4) ALI + AAV-METTL14. Each group received a dose of 50 μl viral solution (either AAV-GFP or AAV-METTL14 with 1012 vg/ml titer)

via intranasal instillation 4 weeks before being challenged with LPS or saline. Mice in the fifth series were grouped as follows: (1) ALI + AAV-GFP + DMSO, (2) ALI + AAV-METTL14 + DMSO, (3)

ALI + AAV-GFP + MCC950, or (4) ALI + AAV-METTL14 + MCC950. Mice in the sixth series were grouped as follows: (1) ALI + AAV-GFP, (2) ALI + AAV-METTL14, (3) ALI + AAV-METTL14 + si-NC, or (4)

ALI + AAV-METTL14 + si-IGF2BP2. The treatments of each group were performed according to the mentioned above. HISTOPATHOLOGICAL ANALYSIS Left lung lobes were fixed in 4% paraformaldehyde for

48 h, dehydrated, embedded in paraffin, and sliced into 5-µm-thick sections. The sections were then stained with hematoxylin and eosin (H&E) according to the manufacturer’s

instructions, to evaluate lung histopathology. The damage to the lung tissues was scored using a previously described semiquantitative scoring system [47]. Images were captured using a

microscope (NIKON Eclipse Ni-U; NIKON, Tokyo, Japan). LUNG WET/DRY RATIO Any blood present on the isolated right lungs was blotted with filter paper before the weights of these lungs were

recorded as wet weight. Then, the lungs were then stored in an incubator at 60 °C for 48 h, following which the weight of the lungs was recorded as dry weight. The lung wet/dry (W/D) ratio

was used to evaluate the degree of pulmonary edema. BRONCHOALVEOLAR LAVAGE At the time of lavage, the mice were anesthetized with an i.p. injection of 1% pentobarbital sodium (50 mg/kg). The

chest cavity was exposed, then the trachea was intubated, and a whole lung lavage was performed by employed sterile PBS (1 mL). The collected lavage fluid was centrifuged at 1000×_g_ for 10

 min at 4 °C, and the cell-free supernatants were harvested and stored at −80◦C for further analysis. The total protein concentration of bronchoalveolar lavage fluid (BALF) was measured

using the BCA Protein Assay Kit (Thermo Fisher Scientific). CELL CULTURE AND TREATMENTS RAW264.7 cells, a mouse macrophage cell line, was obtained from ATCC and cultured in Dulbecco’s

modified Eagle’s medium (Gibco from Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (Gibco) in an incubator at 37 °C and 5% CO2. To establish an NLRP3 inflammasome

activation model in vitro, RAW264.7 cells were stimulated with LPS (1 μg/mL) for 6 h, then treated with nigericin (10 μM, InvivoGen, San Diego, CA, USA) for 30 min. For transient

transfection purpose, cells were seeded at 30–40% confluence and cultured overnight, following which si-METTL14, si-IGF2BP2, and negative control (si-NC) were diluted in Opti-MEM® medium

(Thermo Fisher Scientific, Waltham, MA, USA) and transfected using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After

48 h of transfection, the cells were treated with or without LPS (1 μg/mL) for 6 h. Three siRNA sequences targeting METTL14 and IGF2BP2 were designed and synthesized by RiboBio (Guangzhou,

China) and these were listed in Supplemental Table 1. Specific siRNA with the best knockdown effect was used for further research and in vivo assays. ELISA ANALYSIS AND MYELOPEROXIDASE

ACTIVITY Mice were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg). Blood samples were collected from the retro-orbital sinus after the mice lost

consciousness. Subsequently, blood samples were allowed to clot by leaving them undisturbed at 25 °C for 30 min. The clots were then removed to obtain serum via centrifugation at 1000 × g

for 10 min at 4 °C. Part of the right lung from each mouse was homogenized with ELISA buffer and centrifuged to obtain lung tissue supernatants. Samples of murine serum, lung tissue

supernatants, and cell culture supernatants were used to quantify the concentrations of IL-1β (R&D System, Minneapolis, MN, USA) and IL-18 (R&D System) by using murine ELISA kits,

according to the manufacturer’s instructions. The MPO activity in the lung tissue was assessed by an MPO assay kit (R&D System). RNA M6A DOT BLOT ASSAY Total RNA and poly-A RNA were

isolated from the lung tissue or RAW264.7 macrophages using a RNeasy mini kit (Qiagen, Düsseldorf, Germany) and a Dynabeads® mRNA purification kit (Ambion, Austin, TX, USA), according to the

manufacturer’s instructions. RNA was quantified using a Nanodrop, and equal amounts of RNA were crosslinked onto Hybond-N discs (Cytiva, USA) using a UV crosslinker (Spectroliner, Long

Island, NY, USA). The membrane was quickly washed and blocked using 5% nonfat dry milk in 0.1% phosphate-buffered saline with Tween-20 (PBST) supplemented with RNaseOUT (Thermo Fisher

Scientific). The membrane was incubated overnight at 4 °C with rabbit anti-m6A antibody (1:500, cat#A-1802-100, EpiGentek, Farmingdale, NY, USA), followed by incubation with horseradish

peroxidase (HRP)-conjugated secondary anti-rabbit antibody. Membranes were washed and visualized using an enhanced chemiluminescence detection system. Images were acquired using a ChemiDoc™

Touch Imaging System (Bio-Rad, Berkeley, CA, USA). Finally, the membranes were stained with methylene blue as a loading control. The signal intensity of the dot blot was analyzed using

ImageJ software (NIH, Bethesda, MD, USA). RNA M6A MODIFICATION QUANTIFICATION The levels of m6A in lung tissues and RAW264.7 macrophages were quantified using an EpiQuik m6A RNA Methylation

Quantification Kit (EpiGentek), according to the manufacturer’s recommendations. WESTERN BLOTTING Mouse lung tissues or cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China)

containing a protein inhibitor cocktail (Roche, Mannheim, Baden Württemberg, Germany). The total protein concentration was quantified using a BCA kit (Thermo Fisher Scientific). Samples were

denatured at 100 °C for 10 min and separated on 10–12% SDS-PAGE gels with a molecular weight standard. Proteins from SDS-PAGE gel were transferred onto PVDF membranes (Merck Millipore,

Darmstadt, Germany), blocked using 5% non-fat milk for 2 h, and incubated at 4 °C with the following primary antibodies overnight: METTL3 (1:1000, 15073-1-AP, Proteintech, Wuhan, China),

METTL14 (1:1000, A8530, Abclonal, Boston, MA, USA), METTL16 (1:1000, 17676 S, Cell Signaling Technology, Danvers, MA, USA), WTAP (1:1000, 60188-1-Ig, Proteintech), FTO (1:1000, 45980 S, Cell

Signaling Technology), ALKBH5 (1:1000, 16837-1-AP, Proteintech), NLRP3 (1:1000, AG-20B-0006-C100, AdipoGen, San Diego, CA, USA), Caspase-1 (1:1000, AG-20B-0042-C100, AdipoGen), IL-1β

(1:500, AF-401-NA, R&D System), IGF2BP1 (1:1000, A1517, Abclonal), IGF2BP2 (1:1000, 11601-1-AP, Proteintech), IGF2BP3 (1:1000, A23295, Abclonal). After three washes, the membranes were

incubated with the corresponding HRP-conjugated secondary antibody (1:1000, Abcam, Cambridge, UK) at room temperature for 1 h. Protein bands were detected using ECL and visualized using a

ChemiDoc™ Touch Imaging System (Bio-Rad). The band intensities were analyzed by using ImageJ software. IMMUNOFLUORESCENCE Left lung lobes were fixed with 4% paraformaldehyde for 48 h,

dehydrated, embedded in paraffin, and sectioned into 5-µm slices. After deparaffinated, dehydration, and antigen recovery, sections were incubated in blocking solution (Beyotime) for 1 h at

room temperature and then incubated with primary antibodies overnight at 4 °C, followed by incubation at 25 °C for 1 h with fluorescently labeled secondary antibodies. Nuclei were stained

for 10 min with DAPI. Images from six representative non-overlapping high-power fields (HPFs) of individual mice were taken on a fluorescent microscope (Leica, Wetzlar, Germany) in a blinded

manner. The following antibodies were used: anti-METTL14 (1:200, A8530, Abclonal), anti-CD68 (18985-1-AP, 1:100, Proteintech), anti-F4/80 (18985-1-AP, 1:100, Proteintech), anti-Siglec F

(18985-1-AP, 1:100, Proteintech), and anti-CK7 (16001- 1-AP, 1:100; Proteintech). QUANTITATIVE REAL-TIME RT-PCR Total RNA was extracted using TRIzol reagent (Invitrogen), and subsequently

reverse-transcribed to cDNA according to the instructions of manufacturer of the qPCR transcription kit (EZ Bioscience, Roseville, NM, USA). Quantitative PCR was performed using SYBR Green

Mix (EZ Bioscience) and a CFX96 Real-Time PCR Detection System (Bio-Rad). Target mRNA expression was calculated via the 2-ΔΔCt method using GAPDH as an endogenous control. Primer sequences

are listed in Supplemental Table 2. RNA STABILITY ASSAY RAW264.7 macrophages were cultured in six-well culture plates until they reached 80% confluence. Actinomycin D (Abmole, Houston, TX,

USA) was added at a final concentration of 5 μg/ml. Cells were collected at 0, 0.5, 1, 2, 4, and 6 h. Total RNA was extracted, RT–qPCR was performed as described above, and GAPDH was used as

the loading control for normalization. Then, the RNA half-life was calculated. RNA IMMUNOPRECIPITATION ASSAY RIP was performed according to the manufacturer’s instructions (Merck

Millipore). Briefly, lung tissues of equal weight were mechanically sheared into a single-cell suspension using a homogenizer and resuspended in RIP lysis buffer containing protease

inhibitor and RNase inhibitors. The mixture was centrifuged at 14 000 rpm at 4 °C for 10 min to obtain the supernatant, which was then divided into three fractions: Input, IP, and IgG. Each

fraction was incubated overnight with the corresponding primary antibody at 4 °C, followed by protein A/G bead incubation at room temperature for 30 min. After six washes, the beads were

incubated with 150 μl proteinase K buffer at 55 °C for 30 min. Total RNA was extracted, and the expression of related genes was detected via RT-qPCR. M6A RNA IMMUNOPRECIPITATION ASSAY The

MeRIP assay was performed using a Magna MeRIP m6A Kit (Merck Millipore). Briefly, total RNA was extracted from lung tissues and RAW264.7 macrophages using TRIzol reagent. Approximately 20 μg

of purified RNA was incubated with RNA fragmentation buffer. Then, 1 μg of the fragmented mRNA was used as input, while the remaining RNA was incubated overnight with anti-m6A antibody

(Synaptic Systems, Gottingen, Germany) or anti-IgG antibody in 500 μl of IP buffer at 4 °C. The following procedure was performed in the same manner as that described for the RIP assay. RNA

PULL-DOWN ASSAY An RNA pull-down assay was performed using a Magnetic RNA-Protein Pull-down Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The 3’-end Biotin-TEG

modified-DNA probes against NLRP3 were synthesized by RiboBio. The biotinylated NLRP3 probe (50 pmol) was incubated with streptavidin beads to generate probe-coated beads. Lung tissue

homogenates with probe-coated beads was incubated at 4 °C overnight. After three washes, proteins bound to the beads was boiled and used for the immunoblotting. STATISTICAL ANALYSIS All

sample size information is shown (figure legends). Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad software Inc, San Diego, CA, USA). Quantitative data were assessed

for normality test and presented as the means ± SD. Differences between two normally distributed groups were analyzed using a two-tailed unpaired Student’s _t_ test. Multiple comparisons of

parametric data were performed using one-way ANOVA. Exact _P_ values are indicated in all figures, and statistical significance was set at _P_ < 0.05. DATA AVAILABILITY Data are available

from the corresponding author on reasonable request. REFERENCES * Meyer NJ, Gattinoni L, Calfee CS. Acute respiratory distress syndrome. Lancet. 2021;398:622–37. Article  CAS  PubMed 

PubMed Central  Google Scholar  * Gorman EA, O’Kane CM, McAuley DF. Acute respiratory distress syndrome in adults: diagnosis, outcomes, long-term sequelae, and management. Lancet.

2022;400:1157–70. Article  PubMed  Google Scholar  * Bos LDJ, Ware LB. Acute respiratory distress syndrome: causes, pathophysiology, and phenotypes. Lancet. 2022;400:1145–56. Article  PubMed

  Google Scholar  * Gilroy DW, De Maeyer RPH, Tepper M, O’Brien A, Uddin M, Chen J, et al. Treating exuberant, non-resolving inflammation in the lung; implications for acute respiratory

distress syndrome and COVID-19. Pharmacol Ther. 2021;221:107745. Article  CAS  PubMed  Google Scholar  * Laffey JG, Matthay MA. Fifty years of research in ARDS. Cell-based therapy for acute

respiratory distress syndrome. biology and potential therapeutic value. Am J Respir Crit Care Med. 2017;196:266–73. Article  CAS  PubMed  PubMed Central  Google Scholar  * Xian H, Liu Y,

Rundberg Nilsson A, Gatchalian R, Crother TR, Tourtellotte WG, et al. Metformin inhibition of mitochondrial ATP and DNA synthesis abrogates NLRP3 inflammasome activation and pulmonary

inflammation. Immunity. 2021;54:1463–77.e1411. Article  CAS  PubMed  PubMed Central  Google Scholar  * Paik S, Kim JK, Silwal P, Sasakawa C, Jo EK. An update on the regulatory mechanisms of

NLRP3 inflammasome activation. Cell Mol Immunol. 2021;18:1141–60. Article  CAS  PubMed  PubMed Central  Google Scholar  * Kelley N, Jeltema D, Duan Y, He Y. The NLRP3 inflammasome: an

overview of mechanisms of activation and regulation. Int J Mol Sci. 2019;20:3328. * Swanson KV, Deng M, Ting JP. The NLRP3 inflammasome: molecular activation and regulation to therapeutics.

Nat Rev Immunol. 2019;19:477–89. Article  CAS  PubMed  PubMed Central  Google Scholar  * Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, Speert D, et al. Cutting edge: NF-kappaB

activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J Immunol. 2009;183:787–91. Article  CAS  PubMed  Google Scholar

  * Cornut M, Bourdonnay E, Henry T. Transcriptional regulation of inflammasomes. Int J Mol Sci. 2020;21:8087. * Baker PJ, De Nardo D, Moghaddas F, Tran LS, Bachem A, Nguyen T, et al.

Posttranslational modification as a critical determinant of cytoplasmic innate immune recognition. Physiol Rev. 2017;97:1165–209. Article  CAS  PubMed  Google Scholar  * Moretti J, Blander

JM. Increasing complexity of NLRP3 inflammasome regulation. J Leukoc Biol. 2021;109:561–71. Article  CAS  PubMed  Google Scholar  * Liu Y, Yang D, Liu T, Chen J, Yu J, Yi P.

N6-methyladenosine-mediated gene regulation and therapeutic implications. Trends Mol Med. 2023;29:454–67. Article  CAS  PubMed  Google Scholar  * Zaccara S, Ries RJ, Jaffrey SR. Reading,

writing and erasing mRNA methylation. Nat Rev Mol Cell Biol. 2019;20:608–24. Article  CAS  PubMed  Google Scholar  * Zhao Y, Shi Y, Shen H, Xie W. m(6)A-binding proteins: the emerging

crucial performers in epigenetics. J Hematol Oncol. 2020;13:35. Article  CAS  PubMed  PubMed Central  Google Scholar  * Xu H, Lin C, Yang J, Chen X, Chen Y, Chen J, et al. The role of

N(6)-methyladenosine in inflammatory diseases. Oxid Med Cell Longev. 2022;2022:9744771. Article  PubMed  PubMed Central  Google Scholar  * Peng Z, Gong Y, Wang X, He W, Wu L, Zhang L, et al.

METTL3-m(6)A-Rubicon axis inhibits autophagy in nonalcoholic fatty liver disease. Mol Ther. 2022;30:932–46. Article  CAS  PubMed  Google Scholar  * Zhang J, Song B, Zeng Y, Xu C, Gao L, Guo

Y, et al. m6A modification in inflammatory bowel disease provides new insights into clinical applications. Biomed Pharmacother. 2023;159:114298. Article  CAS  PubMed  Google Scholar  *

Winkler R, Gillis E, Lasman L, Safra M, Geula S, Soyris C, et al. m(6)A modification controls the innate immune response to infection by targeting type I interferons. Nat Immunol.

2019;20:173–82. Article  CAS  PubMed  Google Scholar  * Zhang H, Liu J, Zhou Y, Qu M, Wang Y, Guo K, et al. Neutrophil extracellular traps mediate m(6)A modification and regulates

sepsis-associated acute lung injury by activating ferroptosis in alveolar epithelial cells. Int J Biol Sci. 2022;18:3337–57. Article  CAS  PubMed  PubMed Central  Google Scholar  * Jiao Y,

Zhang T, Zhang C, Ji H, Tong X, Xia R, et al. Exosomal miR-30d-5p of neutrophils induces M1 macrophage polarization and primes macrophage pyroptosis in sepsis-related acute lung injury. Crit

Care. 2021;25:356. Article  PubMed  PubMed Central  Google Scholar  * Huang X, Zhang H, Guo X, Zhu Z, Cai H, Kong X. Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in cancer.

J Hematol Oncol. 2018;11:88. Article  PubMed  PubMed Central  Google Scholar  * Huang H, Weng H, Chen J. m(6)A modification in coding and non-coding RNAs: roles and therapeutic implications

in cancer. Cancer Cell. 2020;37:270–88. Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhu X, Tang H, Yang M, Yin K. N6-methyladenosine in macrophage function: a novel target for

metabolic diseases. Trends Endocrinol Metab. 2023;34:66–84. Article  CAS  PubMed  Google Scholar  * Kumari R, Ranjan P, Suleiman ZG, Goswami SK, Li J, Prasad R, et al. mRNA modifications in

cardiovascular biology and disease: with a focus on m6A modification. Cardiovasc Res. 2022;118:1680–92. Article  CAS  PubMed  Google Scholar  * Luo J, Xu T, Sun K. N6-methyladenosine RNA

modification in inflammation: roles, mechanisms, and applications. Front Cell Dev Biol. 2021;9:670711. Article  PubMed  PubMed Central  Google Scholar  * Weng H, Huang H, Wu H, Qin X, Zhao

BS, Dong L, et al. METTL14 inhibits hematopoietic stem/progenitor differentiation and promotes leukemogenesis via mRNA m(6)A modification. Cell Stem Cell. 2018;22:191–205.e199. Article  CAS

  PubMed  Google Scholar  * Zhang H, Wu D, Wang Y, Guo K, Spencer CB, Ortoga L, et al. METTL3-mediated N6-methyladenosine exacerbates ferroptosis via m6A-IGF2BP2-dependent mitochondrial

metabolic reprogramming in sepsis-induced acute lung injury. Clin Transl Med. 2023;13:e1389. Article  CAS  PubMed  PubMed Central  Google Scholar  * Qu M, Chen Z, Qiu Z, Nan K, Wang Y, Shi

Y, et al. Neutrophil extracellular traps-triggered impaired autophagic flux via METTL3 underlies sepsis-associated acute lung injury. Cell Death Discov. 2022;8:375. Article  CAS  PubMed 

PubMed Central  Google Scholar  * Chen Y, Wu Y, Zhu L, Chen C, Xu S, Tang D, et al. METTL3-mediated N6-methyladenosine modification of Trim59 mRNA protects against sepsis-induced acute

respiratory distress syndrome. Front Immunol. 2022;13:897487. Article  CAS  PubMed  PubMed Central  Google Scholar  * Chen X, Tang J, Shuai W, Meng J, Feng J, Han Z. Macrophage polarization

and its role in the pathogenesis of acute lung injury/acute respiratory distress syndrome. Inflamm Res. 2020;69:883–95. Article  CAS  PubMed  PubMed Central  Google Scholar  * Zaslona Z,

Przybranowski S, Wilke C, van Rooijen N, Teitz-Tennenbaum S, Osterholzer JJ, et al. Resident alveolar macrophages suppress, whereas recruited monocytes promote, allergic lung inflammation in

murine models of asthma. J Immunol. 2014;193:4245–53. Article  CAS  PubMed  Google Scholar  * Han W, Tanjore H, Liu Y, Hunt RP, Gutor SS, Serezani APM, et al. Identification and

characterization of alveolar and recruited lung macrophages during acute lung inflammation. J Immunol. 2023;210:1827–36. Article  CAS  PubMed  Google Scholar  * Li Y, Li J, Yu Q, Ji L, Peng

B. METTL14 regulates microglia/macrophage polarization and NLRP3 inflammasome activation after ischemic stroke by the KAT3B-STING axis. Neurobiol Dis. 2023;185:106253. Article  CAS  PubMed 

Google Scholar  * Zheng Y, Li Y, Ran X, Wang D, Zheng X, Zhang M, et al. Mettl14 mediates the inflammatory response of macrophages in atherosclerosis through the NF-kappaB/IL-6 signaling

pathway. Cell Mol Life Sci. 2022;79:311. Article  CAS  PubMed  PubMed Central  Google Scholar  * Dang W, Tao Y, Xu X, Zhao H, Zou L, Li Y. The role of lung macrophages in acute respiratory

distress syndrome. Inflamm Res. 2022;71:1417–32. Article  CAS  PubMed  PubMed Central  Google Scholar  * Wang X, Ding Y, Li R, Zhang R, Ge X, Gao R, et al. N(6)-methyladenosine of Spi2a

attenuates inflammation and sepsis-associated myocardial dysfunction in mice. Nat Commun. 2023;14:1185. Article  CAS  PubMed  PubMed Central  Google Scholar  * Mangan MSJ, Olhava EJ, Roush

WR, Seidel HM, Glick GD, Latz E. Targeting the NLRP3 inflammasome in inflammatory diseases. Nat Rev Drug Discov. 2018;17:588–606. Article  CAS  PubMed  Google Scholar  * Wann SR, Chi PL,

Huang WC, Cheng CC, Chang YT. Combination therapy of iPSC-derived conditioned medium with ceftriaxone alleviates bacteria-induced lung injury by targeting the NLRP3 inflammasome. J Cell

Physiol. 2022;237:1299–314. Article  CAS  PubMed  Google Scholar  * Xu Q, Ye Y, Wang Z, Zhu H, Li Y, Wang J, et al. NLRP3 knockout protects against lung injury induced by cerebral

ischemia-reperfusion. Oxid Med Cell Longev. 2022;2022:6260102. PubMed  PubMed Central  Google Scholar  * Bakhshi S, Shamsi S. MCC950 in the treatment of NLRP3-mediated inflammatory diseases:

Latest evidence and therapeutic outcomes. Int Immunopharmacol. 2022;106:108595. Article  CAS  PubMed  Google Scholar  * Li H, Guan Y, Liang B, Ding P, Hou X, Wei W, et al. Therapeutic

potential of MCC950, a specific inhibitor of NLRP3 inflammasome. Eur J Pharmacol. 2022;928:175091. Article  CAS  PubMed  Google Scholar  * Meng L, Lin H, Huang X, Weng J, Peng F, Wu S.

METTL14 suppresses pyroptosis and diabetic cardiomyopathy by downregulating TINCR lncRNA. Cell Death Dis. 2022;13:38. Article  CAS  PubMed  PubMed Central  Google Scholar  * Yuan X, Li T,

Shi L, Miao J, Guo Y, Chen Y. Human umbilical cord mesenchymal stem cells deliver exogenous miR-26a-5p via exosomes to inhibit nucleus pulposus cell pyroptosis through METTL14/NLRP3. Mol

Med. 2021;27:91. Article  CAS  PubMed  PubMed Central  Google Scholar  * Huang H, Weng H, Sun W, Qin X, Shi H, Wu H, et al. Recognition of RNA N (6)-methyladenosine by IGF2BP proteins

enhances mRNA stability and translation. Nat Cell Biol. 2018;20:285–95. Article  CAS  PubMed  PubMed Central  Google Scholar  * Cao F, Tian X, Li Z, Lv Y, Han J, Zhuang R, et al. Suppression

of NLRP3 inflammasome by erythropoietin via the EPOR/JAK2/STAT3 pathway contributes to attenuation of acute lung injury in mice. Front Pharmacol. 2020;11:306. Article  CAS  PubMed  PubMed

Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank our colleagues for technical help and stimulating discussion. This work was supported by grants from the National

Natural Science Foundation of China (81870878, 8217102207, 82101348), Guangdong Basic and Applied Basic Research Foundation (2019B151502010, 2022B1515120026, and 2021A1515220117). AUTHOR

INFORMATION Author notes * These authors contributed equally: Fei Cao, Guojun Chen, Yixin Xu. AUTHORS AND AFFILIATIONS * Department of Anesthesiology, State Key Laboratory of Oncology in

South China, Guangdong Provincial Clinical Research Center for Cancer, Sun Yat-sen University Cancer Center, Guangzhou, 510060, P. R. China Fei Cao, Guojun Chen, Yixin Xu, Xintong Wang, 

Xiaole Tang, Wenyu Zhang, Xiong Song, Xiaohua Yang, Weian Zeng & Jingdun Xie Authors * Fei Cao View author publications You can also search for this author inPubMed Google Scholar *

Guojun Chen View author publications You can also search for this author inPubMed Google Scholar * Yixin Xu View author publications You can also search for this author inPubMed Google

Scholar * Xintong Wang View author publications You can also search for this author inPubMed Google Scholar * Xiaole Tang View author publications You can also search for this author

inPubMed Google Scholar * Wenyu Zhang View author publications You can also search for this author inPubMed Google Scholar * Xiong Song View author publications You can also search for this

author inPubMed Google Scholar * Xiaohua Yang View author publications You can also search for this author inPubMed Google Scholar * Weian Zeng View author publications You can also search

for this author inPubMed Google Scholar * Jingdun Xie View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS JX, WZ, and FC conceived and designed

research; FC, GC, YX, and XW performed research; FC, YX, and JX performed writing, review, and revision of the paper; XT analyzed data; WZ, XS, and XY provided technical and material

support. All authors read and approved the final paper. CORRESPONDING AUTHOR Correspondence to Jingdun Xie. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

interests. ETHICAL APPROVAL The animal study was approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University Cancer Center and carried out under the guidelines of

the Guide for the Care and Use of Laboratory Animals of the China National Institutes of Health. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional affiliations. Edited by Sudan He SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLE 1 SUPPLEMENTARY TABLE 2 SUPPLEMENTARY TABLE 3

SUPPLEMENTARY FGURE 1 SUPPLEMENTARY FGURE 2 SUPPLEMENTARY FIGURE LEGENDS ORIGINAL WESTERN BLOTS RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons

Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original

author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the

article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use

is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit

http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Cao, F., Chen, G., Xu, Y. _et al._ METTL14 contributes to acute lung injury by

stabilizing NLRP3 expression in an IGF2BP2-dependent manner. _Cell Death Dis_ 15, 43 (2024). https://doi.org/10.1038/s41419-023-06407-6 Download citation * Received: 16 May 2023 * Revised:

14 December 2023 * Accepted: 21 December 2023 * Published: 13 January 2024 * DOI: https://doi.org/10.1038/s41419-023-06407-6 SHARE THIS ARTICLE Anyone you share the following link with will

be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt

content-sharing initiative