RNA m6A methylation modulates airway inflammation in allergic asthma via PTX3-dependent macrophage homeostasis

N6-methyladenosine (m6A), the most prevalent mRNA modification, has an important function in diverse biological processes. However, the involvement of m6A in allergic asthma and macrophage homeostasis remains largely unknown. Here we show that m6A methyltransferases METTL3 is expressed at a low level in monocyte-derived macrophages from childhood allergic asthma patients. Conditional knockout of Mettl3 in myeloid cells enhances Th2 cell response and aggravates allergic airway inflammation by facilitating M2 macrophage activation. Loss and gain functional studies confirm that METTL3 suppresses M2 macrophage activation partly through PI3K/AKT and JAK/STAT6 signaling. Mechanistically, m6A-sequencing shows that loss of METTL3 impairs the m6A-YTHDF3-dependent degradation of PTX3 mRNA, while higher PTX3 expression positively correlates with asthma severity through promoting M2 macrophage activation. Furthermore, the METTL3/YTHDF3-m6A/PTX3 interactions contribute to autophagy maturation in macrophages by modulating STX17 expression. Collectively, this study highlights the function of m6A in regulating macrophage homeostasis and identifies potential targets in controlling allergic asthma.

in developing countries 1,5 .A better understanding of the mechanisms underlying the pathogenesis of allergic asthma is critical for improving therapeutic outcomes.
Macrophages are the most abundant immune cells in the lung 6 .As important immune effector cells, macrophages display remarkable plasticity and change their physiology in response to environmental cues.Macrophage function or homeostasis is maintained by various regulatory processes, including the crosstalk between activation and autophagy 7 .The imbalance of macrophage homeostasis significantly affects immune-mediated lung injury associated with microbial infection, asthma, and tumorigenesis 8 .Macrophages are generally divided into classically activated (M1) and alternatively activated (M2) macrophages.The M1 subset, induced by IFN-γ/lipopolysaccharide (LPS), is involved in pro-inflammatory responses.In contrast, interleukin-4 (IL-4)/IL-13-induced M2 macrophages contribute to anti-inflammatory activity, wound healing, tissue repair, and allergic inflammation.A recently reported increased activation of M2 macrophages has been suggested to play a crucial role in allergic asthma through activating Th2 cell response [9][10][11][12][13] .However, how macrophage homeostasis is regulated in allergic asthma is less clear.
N 6 -methyladenosine (m 6 A) modification is the most prevalent internal modification in eukaryotic mRNAs, primarily affecting "RRACH" (R = G or A; H = A, C or U) consensus motif 14,15 .Methyltransferase-like 3 (METTL3), METTL14, and WTAP have been demonstrated to act as m 6 A methyltransferases ('writers'), while modified m 6 A is removed by demethylases, 'erasers', such as FTO and ALKBH5 16,17 .m 6 A-binding proteins from the YT521-B homology (YTH) domain family, including YTHDF1/2/3, are known to be the 'readers' recognizing m 6 A. Members of this family modulate the stability and translation of modified mRNAs 16,18,19 .It has been extensively documented that m 6 A modification plays a crucial role in various biological and pathological processes, such as embryonic stem cell maintenance and differentiation 20 , T-cell homeostasis 21 , and tumorigenesis 22 .Accumulating evidence suggests that m 6 A modifications influence the fate and function of mRNA, which may be critical in macrophage homeostasis 23,24 .Recently, Mettl3-deficient mice show increased M1/ M2-like tumor-associated macrophage and regulatory T cell infiltration into tumors 25 , while Mettl3 deficiency in macrophages attenuates their ability to eliminate pathogens and tumors in vivo 26 .Nonetheless, the roles of m 6 A modification and the underlying connection among m 6 A 'writers', 'readers', and 'targets' in macrophage homeostasis and allergic asthma remain to be fully elucidated.
Here, we report a direct correlation between the decreased expression of METTL3 in monocyte-derived macrophages and the severity of childhood allergic asthma.Mettl3-deficient myeloid cells promote Th2 cell response and aggravate airway inflammation in a mouse allergic asthma model by enhancing M2 macrophage activation.m 6 A-seq and MeRIP-qPCR findings indicate that m 6 A /METTL3/ YTHDF3 interactions are involved in the repression of M2 macrophage activation by accelerating the degradation of Pentraxin 3 (PTX3) transcripts, which is widely regarded as a regulator of inflammation and asthma.Thus, our study illustrates that m 6 A RNA modification regulates macrophage homeostasis and may provide therapeutic targets in the treatment of allergic asthma.

Results
Mettl3 deficiency in myeloid cells aggravates airway inflammation in an allergic asthma model m 6 A methyltransferase, METTL3, has been reported to play a key role in the pathophysiology of several diseases by affecting diverse downstream genes 15,25,26 .To investigate the relevance of m 6 A modifications in myeloid cell-mediated allergic asthma, we first crossed Mettl3 fl/fl mice with Lyz2-Cre mice to ablate Mettl3 in the myeloid compartment, including macrophages and neutrophils (Supplementary Fig. 1).Bone marrow-derived macrophages (BMDMs) from Mettl3 fl/fl (WT) and Mettl3 fl/fl Lyz2 Cre+ (Mettl3 KO) mice were subjected to western blotting.BMDMs from the Mettl3 KO mice showed a significant reduction in METTL3 protein levels, consistent with the reported ∼70%-80% deletion efficiency of the Lyz-Cre system 27 .Compared to WT mice, no apparent abnormalities of external morphology were noted in Mettl3 KO mice.Mettl3 KO mice also developed normally and no spontaneous inflammatory pathologies were detected in various organs.We then sensitized WT and Mettl3 KO mice with cockroach extract (CRE) to induce mouse allergic asthma model (Fig. 1a).
The results showed that, compared to PBS control, CREchallenged mice exhibited a significant increase in the recruitment of inflammatory cells to the lungs, with dense peribronchial infiltrates, goblet cell hyperplasia, and mucus secretion.Compared to CREtreated WT mice, the inflammatory response in CRE-treated Mettl3 KO animals was particularly robust, with significantly increased numbers of total inflammatory cells in bronchoalveolar lavage fluid (BALF), including eosinophils (Fig. 1b, c).Furthermore, compared to WT mice, airway hyperresponsiveness (AHR) during the methacholine challenge was exaggerated in CRE-sensitized Mettl3 KO animals (Fig. 1d).Histological examination confirmed the pronounced recruitment of inflammatory cells to the lung tissues in CRE-treated Mettl3 KO mice, detecting dense peribronchial infiltrates, goblet cell hyperplasia, and mucus secretion (Fig. 1e, f), indicating that METTL3 may play a protective role during the pathogenesis of allergic asthma.
Given that neutrophils express high levels of Lyz2 and can be recruited to lung tissues to exert pro-inflammation effects during asthma 2 , we detected that the infiltration of Gr1 + neutrophils in lung tissues tended to be enhanced in CRE-treated Mettl3 KO mice compared with CRE-treated WT mice (Supplementary Fig. 2).To further examine whether the accelerated airway inflammation in Mettl3 KO mice was related to the neutrophils, we depleted the neutrophils in CRE-induced asthma models by injecting anti-Ly6G Ab.Flow cytometry analysis and histological examination confirmed the depletion of neutrophils by anti-Ly6G Ab could not abrogate the increased airway inflammation phenotype of Mettl3 KO mice (Supplementary Fig. 2).Thus, these findings indicated that the function of METTL3 in allergic airway inflammation was not dependent on neutrophils.

Low METTL3 expression in monocyte-derived macrophages from children with allergic asthma is associated with disease severity
To investigate the expression pattern of METTL3 in allergic asthma, we first analyzed microarray datasets from 5 human asthma patients and 5 healthy controls from the GEO database (GSE27876).The results showed that the expression of known m 6 A writers (i.e., METTL3 and METTL14), erasers (i.e., FTO and ALKBH5), and readers (i.e., YTHDF3) was moderately downregulated in peripheral blood mononuclear cells (PBMCs) of human asthma sufferers (Supplementary Fig. 3a).We next verified that, compared to 50 normal controls, the abundance of METTL3, METTL14, and YTHDF3 was significantly decreased in PBMCs derived from 55 childhood allergic asthma patients, recruited from the Children's Hospital of Fudan University (Fig. 1g and Supplementary Fig. 3b).Similarly, METTL3 expression was lower in primary monocytederived macrophages of childhood allergic asthma patients than in normal controls (Fig. 1h and Supplementary Fig. 3c).
These observations led us to hypothesize that METTL3 may be associated with the severity of childhood allergic asthma.Thus, the ability of monocyte-derived macrophages METTL3 abundance to differentiate childhood asthma patients from healthy participants was firstly assessed by receiver operating characteristic (ROC) curve analysis, yielding area under the curve (AUC) values of 0.79 (Fig. 1i).Moreover, we explored the correlation between monocyte-derived macrophage METTL3 levels and disease severity among patients with asthma.A negative correlation was observed between METTL3 expression and blood eosinophil numbers, and fraction of exhaled nitric oxide (FeNO) levels, confirming the known eosinophilic airway inflammation in allergic asthma patients.In contrast, low levels of METTL3 were associated with the worse pulmonary function (percent predicted forced expiratory volume in 1 s, %FEV 1 ) in patients (Fig. 1j).Together, these data suggest a possible association between the severity of childhood allergic asthma and a decreased METTL3 expression in monocyte-derived macrophages.
METTL3-deficient macrophages are more susceptible to M2 activation through the PI3K/AKT and JAK/STAT6 signaling, thereby accelerating allergic airway inflammation To further confirm whether the role of METTL3 in allergic airway inflammation is due to macrophage-intrinsic effect, we depleted macrophages in vivo using clodronate-containing liposomes (CLs) 28,29 .Flow cytometry analysis confirmed a significant decrease in the percentage of alveolar macrophages in BALF from Mettl3 KO mice treated with CLs, compared to controls (PBS)-treated Mettl3 KO mice (Fig. 2a).In addition, the depletion of macrophages completely reversed the susceptibility of Mettl3 KO mice to allergic airway inflammation, compared with wild-type animals (Fig. 2b-d), suggesting that the vital role of METTL3 in airway inflammation is dependent on macrophages.
In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway gene set data was analyzed in METTL3-deficient macrophages.RNA-seq data indicated that a small number of differentially expressed genes were associated with Phosphatidylinositol 3kinase-AKT (PI3K/AKT) signaling, Janus kinase-signal transducer, and activator of transcription (JAK/STAT) signaling, and the Autophagy pathway (Fig. 2h).It is well-known that the PI3K/AKT and JAK/STAT6signaling pathways play a crucial role in promoting M2 macrophage activation 30,31 .To further explore whether the PI3K/AKT and JAK/ STAT6 signaling pathways were necessary for METTL3-mediated M2 activation, we performed immunoblots, analyzing BMDMs from WT and Mettl3 KO mice.As expected, upon IL-4 stimulation, the phosphorylation levels of AKT and STAT6 in Mettl3-deficient BMDMs were markedly increased.However, the total levels of these proteins were comparable, irrespective of whether BMDMs were derived from Mettl3-deficient or WT animals (Fig. 2i).A similar enhanced phosphorylation of AKT and STAT6 proteins was noted in human THP1derived macrophages after METTL3 knockdown, whereas their phosphorylation were reduced in METTL3-overexpressing cells (Supplementary Fig. 5d).Furthermore, the rescue experiments demonstrated that Mettl3 deficiency enhanced M2-associated genes levels, whereas the inhibition of AKT or STAT6 phosphorylation levels eliminated this effect (Supplementary Fig. 5e).Notably, in accordance with a previously published study 26 , we also found that METTL3 deficiency inhibited M1 macrophage activation through negatively regulating NF-κB signaling (Supplementary Fig. 6).Taken together, these data indicate a vital role for METTL3 in balancing the equilibrium between M1/ M2 macrophage activation by repressing M2 activation via the PI3K/ AKT and JAK/STAT6 signaling.

Mettl3 depletion enhances Th2 cell response in allergic airway inflammation by facilitating M2 macrophage activation
Previous work has demonstrated the importance of increased M2 macrophage activation in the progression of allergic asthma 11,12 .To further investigate whether Mettl3 depletion exacerbated allergic airway inflammation through modulating M2 macrophage activation, we purified alveolar macrophages from lung tissues.The mRNAs and protein levels of M2-associated genes were significantly elevated in alveolar macrophages from CRE-challenged Mettl3 KO mice, compared to CRE-challenged WT animals (Fig. 3a, b).Furthermore, flow cytometry analysis demonstrated that alveolar macrophages from CREchallenged Mettl3 KO mice expressed more CD206 than the same cells from CRE-challenged WT mice (Fig. 3c, d).In accordance with the flow cytometry data, immunofluorescence staining also revealed that the number of M2 macrophages (F4/80 + CD206 + ) increased significantly in the lung tissues of CRE-treated Mettl3 KO mice (Fig. 3e).Thus, these data demonstrated that METTL3 deficiency significantly promoted M2 macrophage activation during allergic airway inflammation.
Allergic asthma has been generally considered a Th2 cellmediated chronic immune response, while Th2 cells produce effector cytokines such as IL-4 and IL-13 to mediate respiratory symptoms.More importantly, accumulating evidence reveals that in allergic asthma, M2 macrophages release high levels of chemokines, including CCL-17 and CCL-22, resulting in the recruitment of Th2 cells and amplification of polarized Th2 cell responses in the bronchial tissue 9,32 .To further determine whether METTL3 depletion promotes Th2 responses in allergic inflammation via M2 macrophage activation, the levels of M2-associated chemokines (i.e., Ccl-17, and Ccl-22) in alveolar macrophages were tested.We found that the mRNA levels of Ccl-17 and Ccl-22 were significantly elevated in alveolar macrophages from CREchallenged Mettl3 KO mice (Fig. 3f).Furthermore, compared to WT mice, the mRNA and protein levels of Th2 cell-associated cytokines (i.e., IL-4, and IL-13) were markedly increased in lung homogenates from CRE-challenged Mettl3 KO mice, whereas the Th1 cell-associated cytokine (ifng) showed downregulated expression and the Th17 cellassociated cytokine (Il-17a) had no difference (Fig. 3g, h).Lastly, the data demonstrated that IL-4-producing Th2 cells were also enhanced in the mediastinal lymph nodes (MLNs) from CRE-challenged Mettl3 KO mice, while IFN-gamma-producing Th1 cells were comparatively reduced (Fig. 3i, j).Collectively, the above findings suggest that METTL3 deficiency promotes Th2 responses and accelerates allergic airway inflammation via enhancing M2 macrophage activation.f Inflammation and PAS scores were quantified (n = 5 animals).RT-qPCR analysis of the transcripts levels of METTL3 in PBMCs (g) and monocyte-derived macrophages (h) from 55 childhood allergic asthma and 50 healthy controls, respectively.The detailed minimum, median, maximum, 25th, 75th percentile (box), and 5th and 95th percentile (whiskers) of box plots were provided in the Source data file.i Receiver operating characteristic (ROC) curve analysis of monocyte-derived macrophages METTL3 levels in the 55 childhood asthma population.j Spearman correlation (two-sided) analysis of monocyte-derived macrophages METTL3 expression, blood eosinophils number, FeNO, and %FEV 1 levels in 55 childhood asthma.Statistical analysis of the data was performed using two-sided unpaired t test (a), Mann-Whitney test (g, h), 1-way ANOVA (b, f), and 2-way ANOVA (c, d) followed by Tukey's multiple comparison tests.Data are presented as means ± SEM from one of three independent experiments.*P < 0.05, ***P < 0.001, ****P < 0.0001; n.s, not significant.

PTX3 is a direct target of METTL3
To identify critical downstream targets of METTL3-mediated m 6 A modification in macrophages, we mapped m 6 A methylomes of METTL3-deficient and control THP1-derived macrophages by m 6 A sequencing (m 6 A-seq) and RNA-seq.Of the 724 m 6 A peaks showing pronounced changes after METTL3-depletion, the vast majority (668, 93.3%) exhibited a significant decrease in m 6 A abundance (Supplementary Fig. 7a).The previously reported consensus "RRACH" motif (R = G or A; H = A, C or U) was identified in both control and METTL3-deficient cells (Fig. 4a).In addition, we detected that the density of m 6 A was markedly enriched near stop codons (Fig. 4b), consistent with previous observations 15,33 .Meanwhile, a similar pattern of total and common m 6 A distribution was seen in control and METTL3deficient macrophages, with m 6 A peaks being particularly abundant in the vicinity of CDS and 3ʹUTR regions (Fig. 4c).We then analyzed the frequency and distribution of m 6 A modifications within differentially expressed mRNAs, selecting m 6 A peaks and mRNA abundance with > 1.2-fold change, P < 0.05.There were 152 hypo-methylated m 6 A peaks whose mRNA transcripts were either significantly downregulated (88, Hypo-down) or upregulated (64, Hypo-up) in METTL3-deficient macrophages.In contrast with the high number of hypo-methylated m 6 A peaks, only 26 hyper-methylated m 6 A peaks were detected.Half of these were downregulated (13 Hyper-down), while the other half showed upregulation (13 Hyper-up) (Fig. 4d).Considering the role of METTL3 in the m 6 A 'writers' complex, up-regulated mRNAs transcripts carrying hypo-methylated m 6 A peaks in METTL3-deficient macrophages were the likely potential downstream targets of METTL3.
Next, we characterize potential targets involved in METTL3regulated M2 activation.The 64 m 6 A-Hypo-up genes were intersected with 28 M2 activation-associated genes with known regulatory functions (listed in Supplementary Table 1).This comparison identified only three overlapping genes, namely pentraxin-3 (PTX3), C-C Motif Chemokine Ligand 13 (CCL13), and RIP-Like Protein Kinase 3 (RIPK3) (Fig. 4e).Among these three, PTX3 has been recently reported to be a positive regulator of M2 macrophage activation in the development of inflammatory diseases 34,35 .Interestingly, we noted that PTX3 levels exhibited the inverse correlation with METTL3 in monocyte-derived macrophages from childhood allergic asthma patients (Fig. 4f).Meanwhile, the m 6 A-seq data identified that the PTX3 m 6 A level was markedly enriched in control cells compared to the METTL3-deficient cells (Fig. 4g), while PTX3 harbored two m 6 A sites in its 3' UTR (AGA 1333 CT and AGA 1406 CA, respectively).Subsequently, the SELECT analysis demonstrated that the relative amount of qPCR products targeting the AGA 1333 CT site was significantly enhanced in the METTL3deficient macrophages compared to the control, whereas no significant difference was observed in the amplification of the AGA 1406 CA site, implying the AGA 1333 CT site could be methylated by METTL3 (Fig. 4h and Supplementary Fig. 7b).Furthermore, based on the AGA 1333 CT site in PTX3 transcripts, we performed MeRIP assays and confirmed that the enrichment m 6 A level of PTX3 transcripts was significantly downregulated in METTL3-deficient macrophages (Fig. 4i).In addition, METTL3 deficiency in both mouse BMDMs and human THP1derived macrophages led to noticeably elevated PTX3 mRNA and protein levels after IL-4 stimulation, whereas overexpression of METTL3 resulted in a remarkable downregulation (Fig. 4j, k, and Supplementary Fig. 8), indicating that PTX3 is a target of METTL3.
To further confirm that the regulation of PTX3 by METTL3 relies on the methylation of mRNA transcripts, we performed luciferase reporter assays with the PTX3 3'UTR constructs containing wild-type or mutant m 6 A sites (m 6 A modified by A 1333 to T substitution).METTL3 depletion in THP1-derived macrophages significantly enhanced the luciferase activity of the reporter construct carrying the PTX3 3'UTR.This increase was abrogated when the putative m 6 A sites were mutated (Fig. 4l), suggesting that the downregulation of PTX3 expression by METTL3 was dependent on m 6 A modification of the 3ʹUTR within PTX3.
It is well-known that m 6 A modification of mRNA transcripts affects mRNA stability 18,22,36 .To investigate whether METTL3 regulated PTX3 expression by this mechanism, we treated WT or Mettl3 KO BMDMs with the transcription inhibitor actinomycin D and followed the decay of PTX3 transcripts.Mettl3 depletion in these experiments markedly increased PTX3 mRNA stability (Fig. 4m).Collectively, these findings imply that METTL3 enhances the m 6 A modification of the 3'UTR of PTX3, leading to the instability and degradation of PTX3 transcripts.

Disruption of PTX3 attenuates METTL3 depletion-induced M2 macrophage activation and allergic airway inflammation
Next, we explored whether the effect of METTL3 on M2 macrophage activation was mediated via PTX3.The levels of M2-associated markers were significantly downregulated in Ptx3 knockdown BMDMs.This coincided with a notable reduction of PI3K/AKT and JAK/ STAT6 signaling in IL-4-treated M2 macrophages (Fig. 5a, b).Similar observations were made in THP1-derived macrophages after PTX3 knockdown (Supplementary Fig. 9), implying that the downregulation of PTX3 inhibited M2 macrophage activation.Additionally, the data detected that the enhanced effect of Mettl3 deficiency on M2 macrophage activation was markedly abolished by Ptx3 knockdown (Fig. 5c).These results suggest that the upregulation of PTX3 is responsible for the preferential M2 macrophage activation seen in METTL3-deficient macrophages.
Thus, we hypothesized that the overexpression of PTX3 may be responsible for the enhanced allergic inflammation in Mettl3 deficient mice.To test this, we first depleted macrophages in vivo using clodronate-containing liposomes (CLs).In contrast with our findings in Mettl3 KO mice, the data detected that compared to shCtrl-infected mice, Ptx3 knockdown in vivo noticeably alleviated the CRE-induced allergic airway inflammation.However, we found that, through the depletion of macrophages, there was no significant difference in airway inflammation between shPtx3-treated mice (shPtx3 CLs) and shCtrl-mice (shCtrl CLs) (Supplementary Fig. 10), suggesting that the role of Ptx3 knockdown in allergic airway inflammation may be due to macrophages.Subsequently, rescue experiments in vivo were performed by injecting shPtx3 or control (shCtrl) lentiviruses into CREsensitized mice.As expected, the increased concentration of serum PTX3 in CRE-treated Mettl3 KO mice was largely reversed by the shRNA-mediated knockdown of Ptx3 (Fig. 5d).In addition, BALF from shPtx3-infected Mettl3 KO mice contained a remarkably lower number of total inflammatory cells than those from shCtrl-infected Mettl3 KO animals.A prominent reduction in the number of eosinophils was also seen in shPtx3-infected animals (Fig. 5e, f).AHR, peribronchial inflammation, goblet cell hyperplasia, and mucus secretion seen in Mettl3 KO mice were substantially diminished after infection with the shPtx3 lentiviruses (Fig. 5g-i).Overall, these data confirmed that reducing Ptx3 could attenuate airway inflammatory responses in Mettl3 deficiency animals.
Importantly, we also found that by inhibiting the expression of Ptx3, the abundance of M2 activation-associated markers could be substantially reduced in alveolar macrophages (Fig. 5j, k).Moreover, Fig. 2 | METTL3-deficient macrophages are more susceptible to M2 activation through the PI3K/AKT and JAK/STAT6 signaling, thereby accelerating allergic airway inflammation.a CLs-liposome was i.p. administered in the clodronate group and PBS was administered in the control group.Flow cytometry analysis of the efficiency of macrophage depletion in BALF from Mettl3 KO mice by clodronate treatment (n = 5 animals).b Total and (c) differential BALF cell numbers from experimental asthmatic animals were analyzed by flow cytometry (n = 5 animals).d Histopathological changes in the lung tissues were examined by H&E-and PASstaining.Scale bars: 200 μm and 100 μm, respectively.Inflammation and PAS scores were quantified (low panel) (n = 5 animals).e Heatmap illustrating the upregulated expression of M2 activation-associated genes in human THP1-derived macrophages transfected with METTL3 siRNA pools (200 nM), compared with negative control (NC) cells.f RT-qPCR analysis of M1 and M2-associated markers expression in WT and Mettl3 KO mice BMDMs (n = 3 animals) stimulated with LPS or IL-4, respectively.g ELISA shows the levels of TNF and IL-10 secretion in WT and Mettl3 KO mice BMDMs (n = 5 animals).h KEGG enrichment analysis shows the top pathways enriched in METTL3-deficient macrophages.i Western blot showing levels of AKT phosphorylation (p-AKT), AKT, STAT6, and p-STAT6 in WT and Mettl3 KO BMDMs (n = 3 animals), following IL-4 stimulation.Statistical analysis of the data was performed using two-sided unpaired t test (a, f, g, h), 1-way ANOVA (b, d), and 2-way ANOVA (c, i) followed by either Tukey's or Sidak's multiple comparison tests.Data are presented as means ± SEM from one of three independent experiments.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s, not significant.
flow cytometry analysis showed that alveolar macrophages from shPtx3-infected Mettl3 KO mice expressed lower CD206 levels than those from shCtrl-infected Mettl3 KO mice (Fig. 5l).Immunofluorescence staining detected a similar decline in the number of M2 macrophages (F4/80 + CD206 + ) in the lung tissues from shPtx3-treated Mettl3 KO mice, compared to shCtrl-treated Mettl3 KO animals (Fig. 5m).Taken together, the above data strongly suggest that METTL3 inhibits M2 macrophage activation and alleviates airway inflammation in allergic asthma via repressing PTX3 expression.

Loss of METTL3 impairs the YTHDF3-mediated degradation of PTX3 mRNA
Recent studies have illustrated the importance of the family of YTHDF proteins, m 6 A 'readers', in the regulation of physiological processes and various diseases 16,18 .Interestingly, during the analysis of downregulated genes in METTL3-deficient macrophages, we noted that the mRNA level of YTHDF3 was robustly decreased in METTL3-deficient macrophages (Fig. 6a).Consistent with the findings in RNA-seq, YTHDF3 protein levels were suppressed in both Mettl3 KO BMDMs and METTL3-deficient THP1-derived macrophages (Fig. 6b).It was notable that YTHDF3 abundance was also significantly reduced in the monocyte-derived macrophages from childhood allergic asthma (Fig. 6c).YTHDF3 has been shown to play a key role in mediating the degradation and translation of m 6 A-modified mRNAs 19,37,38 .To further verify whether YTHDF3 was involved in the effect of METTL3 on the M2 macrophage activation, we knocked down the expression of YTHDF3 in human THP1-derived macrophages.The results revealed that the loss of YTHDF3 enhanced the expression of M2 activation-associated genes, accompanied by increased phosphorylation of STAT6 and AKT (Fig. 6d, e).These findings were consistent with observations made in METTL3-deficient macrophages.In addition, rescue experiments demonstrated that enforced expression of METTL3 inhibited M2associated genes levels, whereas knockdown of YTHDF3 abolished this effect (Fig. 6f).Therefore, the regulatory function of METTL3 on M2 macrophage activation seems dependent on the presence of YTHDF3.
Moreover, to confirm whether METTL3 represses PTX3 expression through YTHDF3-mediated m 6 A modification, we performed RIP-qPCR assays targeting the m 6 A site in PTX3 transcripts.As expected, PTX3 m 6 A transcripts were remarkably enriched in YTHDF3-pull precipitates, while this relative enrichment was significantly decreased in METTL3-deficient macrophages (Fig. 6g).Furthermore, similar to our findings in METTL3-depleted macrophages, elevated PTX3 mRNA and protein levels were also found in human YTHDF3 knockdown macrophages (Fig. 6h, i).Rescue experiments revealed that the decreased PTX3 levels seen in METTL3-overexpressing human macrophages could be largely reversed by the knockdown of YTHDF3 (Supplementary Fig. 11).mRNA decay assays demonstrated that the degradation of PTX3 transcripts was appreciably attenuated in YTHDF3-deficient macrophages (Fig. 6j).In addition, luciferase reporter assays showed that YTHDF3 knockdown significantly increased the activity of a luciferase construct containing the PTX3 3'UTR, but such increment was largely impaired in PTX3 mutant 3ʹUTR (Fig. 6k), confirming the m 6 A-dependent regulation of PTX3 expression.Overall, these findings indicate that the METTL3/YTHDF3 m 6 A axis directly downregulates PTX3 expression in macrophages by reducing its mRNA stability.

METTL3/YTHDF3-m 6 A/PTX3/STX17 axis controls autophagy maturation in macrophages
To further explore the roles of PTX3 in macrophage functions, we performed RNA-seq in PTX3 knockdown macrophages stimulated with IL-4.As expected, KEGG pathway analysis revealed that the differently expressed transcripts were enriched in PI3K/AKT and JAK/STAT signaling pathways.Interestingly, genes involved in the autophagy pathway were also enriched in PTX3-deficient macrophages, consistent with previous findings in METTL3-depleted cells.Specifically, the data showed that genes enriched in autophagy were associated with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes (Fig. 7a), which are essential for the fusion between autophagosome and lysosome (also called autophagy maturation), and autophagosome degradation 39,40 .Since autophagy is crucial to promote M2 macrophage activation 41,42 , we speculated that PTX3 was required for autophagy-mediated macrophage homeostasis.To test this, we performed transmission electron microscopy.The data showed that stimulating with rapamycin and IL-4, Ptx3 knockdown in macrophages exhibited the up-regulated number of autophagosomes, suggesting either an increased autophagy flux or a defect of autophagosome degradation (Fig. 7b).We then transduced an adenovirus plasmid encoding mRFP-GFP-LC3 into control and Ptx3-deficient BMDMs.After inducing autophagy, the GFP was degraded in control cells but the majority of LC3 dots in Ptx3-deficient cells remained RFP and GFP double-positive, indicating the accumulation of autophagosomes without degradation in Ptx3-deficient cells (Fig. 7c).In contrast, the number of autophagosomes was significantly reduced in both Mettl3 KO BMDMs and YTHDF3 knockdown THP1-derived macrophages (Fig. 7d, e).Additionally, as compared with the control cells, the majority of LC3 dots in both Mettl3 KO BMDMs and YTHDF3 knockdown THP1-derived macrophages just remained RFP-positive (Fig. 7f, g), implying the marked degradation of autophagosomes in these cells.Collectively, these findings imply that the METTL3/YTHDF3-m 6 A/PTX3 axis in macrophages exerts regulatory effects on autophagy maturation.
More importantly, the RNA-seq analysis identified that syntaxin 17 (STX17), a vital component of SNARE complexes, was among the top down-regulated genes in PTX3 deficient macrophages (Fig. 7h).PTX3 knockdown in both BMDMs and THP1-derived macrophages strongly decreased STX17 mRNA and protein expression (Fig. 7i and Supplementary Fig. 12a).By contrast, the STX17 levels were enhanced in both Mettl3 KO BMDMs and YTHDF3 knockdown THP1-derived macrophages (Fig. 7j, k), indicating that METTL3/YTHDF3-m 6 A/PTX3 axis regulates the STX17 expression.Next, to explore whether the PTX3/STX17 axis regulated autophagy maturation in macrophages, Ptx3 knockdown and control BMDMs were treated with rapamycin and the level of LC3-II was measured.Expectedly, STX17 expression was significantly reduced in Ptx3-deficient BMDMs, although, with time, LC3-II was eventually degraded in control but not Ptx3-deficient cells (Fig. 7l).Similar findings were obtained in PTX3-deficient THP1-derived macrophages (Supplementary Fig. 12b), implying a defect of autophagy maturation-mediated autophagosome degradation in Ptx3-deficient macrophages.Notably, our rescue experiments demonstrated that the downregulated autophagosomes seen in Mettl3 KO BMDMs could be largely reversed by the knockdown of Stx17 (Supplementary Fig. 12c).Thus, the data above suggest that the regulatory effect of the METTL3/YTHDF3-m 6 A/PTX3 axis on macrophage homeostasis may be partially related to its role in autophagy maturation via an STX17-dependent manner.
PTX3 expression is enhanced in both monocyte-derived macrophages and plasma from children with allergic asthma PTX3 has been studied as an emerging biomarker reflecting tissue injuries and inflammation, such as lung carcinoma and heart in alveolar macrophages purified from CRE-induced asthma models were determined, using RT-qPCR and Western blot, respectively.c Flow cytometry analysis of M2 macrophage subpopulation in BALF from experimental models.d The MFI of M2 macrophages was calculated (n = 4 animals).e Representative composite fluorescent images showing the number of M2 macrophages (F4/80 + CD206 + ) in lung tissues from experimental models.Total macrophages, M2 macrophages, and nuclei were labeled with F4/80 (red), CD206 (green), and DAPI (blue), respectively.Scale bars: 25 μm.Quantification of the CD206 intensity ratio in macrophages was performed by Image J (bottom) (n = 3 animals).f RT-qPCR showing up-regulated M2-associated chemokines in alveolar macrophages purified from experimental models (n = 4 animals for PBS groups and n = 5 animals for CRE groups).The levels of Th1, Th2, and Th17 cell-associated cytokines in lung homogenates were detected by RT-qPCR (g) (n = 4 animals for PBS groups and n = 5 animals for CRE groups) and ELISA (h) (n = 5 animals), respectively.i, j Flow cytometry analysis of the frequency of CD4 + IL-4 + Th2 cells and CD4 + IFN-gamma + Th1 cells in MLNs from mice (n = 5 animals).Statistical analysis of the data was performed using two-sided unpaired t test with Welch's correction (h left) or not (a, h right, j), 1-way ANOVA (b, d, e), and 2-way ANOVA (f, g) followed by Tukey's multiple comparison tests.Data are presented as means ± SEM from one of three independent experiments.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s, not significant.failure 43,44 .To further elucidate the potential importance of PTX3 in allergic asthma, we evaluated its expression in childhood allergic asthma and healthy controls.In line with observations in the mouse model, both the PTX3 mRNA levels in monocyte-derived macrophages and PTX3 circulating levels in plasma were higher in childhood allergic asthma than in healthy participants (Fig. 8a, b).Importantly, STX17, as a key target of PTX3-mediated macrophage homeostasis, was also more abundant in monocyte-derived macrophages isolated from asthma patients (Fig. 8c).ROC curve analysis, yielding AUC values of 0.69, indicated that PTX3 circulating levels in plasma may be useful for diagnosing childhood allergic asthma (Fig. 8d).Additionally, PTX3 circulating levels showed a positive correlation with blood eosinophils numbers and FeNO levels in childhood allergic asthma (Fig. 8e).Taken together, these findings imply that PTX3 may be a potential biomarker for the diagnosis and assessment of childhood allergic asthma.Discussion m 6 A mRNA modification emerges as an important regulator of diverse biological processes involved in various disease states.In this study, we demonstrated that m 6 A modification could regulate the progression of allergic asthma by influencing macrophage homeostasis.In brief, METTL3 was lowly expressed in monocyte-derived macrophages from childhood allergic asthma, while an inverse correlation between METTL3 expression and disease severity was identified.Conditional knockout of Mettl3 in myeloid cells enhanced Th2 cell response and aggravated allergic airway inflammation by facilitating M2 macrophage activation.Gain and loss of function studies revealed that METTL3 acted as a negative regulator of M2 macrophages, partly through the suppression of PI3K/AKT and JAK/STAT6 signaling.Mechanistically, using m 6 A-seq and RNA-seq, we verified that METTL3 inhibited M2 macrophage activation by promoting the degradation of PTX3 mRNA in a YTHDF3-mediated, m 6 A-dependent manner.Silencing Ptx3 significantly alleviated allergic inflammation in Mettl3 KO mice, whereas higher PTX3 expression in plasma was positively associated with airway inflammation in childhood allergic asthma.Notably, the regulatory effect of the METTL3/YTHDF3-m 6 A/PTX3 axis on macrophage homeostasis was also extended to its role in autophagy maturation via regulating STX17 expression.Collectively, this study highlights the critical role of METTL3 deficiency in the pathogenesis of allergic asthma airway inflammation, as featured by promoting M2 macrophage activation and enhancing Th2 response, and uncovers a signaling axis involving METTL3/YTHDF3-m 6 A/PTX3/STX17 in macrophage activation and autophagy maturation (Fig. 8f).
Altered m 6 A modification has been reported in various diseases, including inflammatory conditions.Our work demonstrated an association between decreased METTL3 levels in monocyte-derived macrophages and the severity of childhood allergic asthma.Combined with neutrophils and macrophage depletion in vivo, we confirmed that Mettl3 deficiency in myeloid cells aggravated airway inflammation via macrophages.An increasing number of studies demonstrated a positive correlation between asthma severity and elevated M2 macrophage numbers [10][11][12][13] .Notably, recent evidence suggests that METTL3 could facilitate or repress the activation of M1 macrophages depending on the local immune microenvironment 25,26,45 , indicating that m 6 A modification may play important role in macrophage homeostasis.In this study, through a combination of RNA-seq, flow cytometry, and immunofluorescence, we observed that M2 activation-associated markers were significantly enhanced in Mettl3-deficient macrophages and lung tissues, accompanied by the activation of PI3K/AKT and JAK/ STAT6 signaling.Allergic asthma is often considered a Th2 cellmediated chronic immune response, while M2 macrophages could release chemokines such as CCL-17 and CCL-22 to recruit the Th2 cells and amplify Th2 cell responses in the bronchial tissue 32 .Here, we revealed that Mettl3 deficiency in mice highly promoted Th2 cell responses in lung tissues via facilitating M2 macrophage activation, implying the importance of METTL3 in allergic airway inflammation.
MeRIP-seq data revealed that the 3ʹUTR regions of PTX3 transcripts contained m 6 A modifications, and eliminating these retarded the degradation of PTX3 mRNA.PTX3, a member of the pentraxin family, is primarily produced by dendritic cells, macrophages, and endothelial cells in response to inflammation.It can have potentially conflicting roles in the regulation of inflammation, antimicrobial resistance, and disease pathogenesis, depending on tissue context, cellular source, and abundance 34,44,46 .PTX3 affects inflammation through multiple mechanisms, such as interaction with complement components, Fcγ receptors, and pathogenic moieties in microbes 47 .The current study demonstrated that PTX3 depletion significantly suppressed M2 macrophage activation through PI3K/AKT and JAK/ STAT6 signaling, consistent with previous reports documenting that PTX3 reduces M1 and increases M2 macrophage activation 34,35 .Furthermore, it has been suggested that plasma PTX3 levels could act as a biomarker in various lung diseases, such as lung carcinoma 43 , chronic obstructive pulmonary disease (COPD) 48 , and acute lung injury (ALI) 49 , while sputum PTX3 levels represent a candidate biomarker for the evaluation of airway inflammation and remodeling in children with asthma 50 .Interestingly, a recent study identified that exogenous PTX3 could exacerbate asthma, by promoting eosinophil infiltration into the lung 51 .However, another independent study found that Ptx3 deficiency in an OVA-induced mouse asthma model resulted in augmented AHR, mucus production, and IL-17-dominant airway inflammation 52 .In this study, we detected elevated levels of serum PTX3 in CRE-treated Mettl3 KO mice.We observed that Ptx3 deficiency markedly attenuated Mettl3 knockout-induced airway inflammation in allergic asthma by suppressing M2 macrophage activation, but the effect of PTX3 on the other cells in vivo can not be excluded.Noticeably, our data also uncovered that plasma levels of PTX3 were higher in childhood allergic asthma patients, showing a positive correlation with disease severity.These findings identify PTX3 as a key regulator of M2 macrophage activation and a potential biomarker in allergic asthma.m 6 A modification accomplishes its biological function primarily by recruiting various RNA-binding proteins, which subsequently direct multi-protein complexes to selectively bind to m 6 A-containing transcripts.We detected a robust decrease of YTHDF3 levels in METTL3deficient macrophages, and in monocyte-derived macrophages from children with allergic asthma.It appears that METTL3 regulates M2 macrophage activation in a YTHDF3-dependent manner.Cytoplasmic 'readers', such as YTHDF1 and YTHDF2, were demonstrated to modulate the degradation and translation of m 6 A-modified mRNAs 16,18 .Although the cytoplasmic m 6 A 'reader' YTHDF3 has been reported to e Venn diagram showing the number of overlapping genes between m 6 A-Hypoupregulated genes in METTL3-deficient macrophages and M2 activation-associated functional genes.f Spearman correlation (two-sided) between METTL3 and PTX3 mRNA levels in monocyte-derived macrophages from 55 childhood allergic asthma populations.g Integrative Genomics Viewer (IGV) showing the m 6 A abundance on PTX3 mRNA transcripts in METTL3 deficiency and control macrophages as detected by m 6 A-seq.h SELECT assay showing the relative amount of qPCR products targeting the AGA 1333 CT site on PTX3 3ʹUTR in THP1-derived macrophages transfected with METTL3 or control siRNA (n = 3 cells).i MeRIP-qPCR assay validated the enrichment of m 6 A-modified PTX3 transcripts in METTL3-deficient THP1-derived macrophages (n = 4 cells).j RT-qPCR (n = 4 animals) and ELISA (n = 6 animals for WT group and n = 4 animals for KO group) showing increased PTX3 mRNA and secretion levels in Mettl3 KO BMDMs.k Protein levels of PTX3 in WT and Mettl3 KO BMDMs were detected by Western blot (n = 3 animals).l Luciferase reporter and mutagenesis assays.Wild-type (WT) or mutant (m 6 A 1333 was replaced by T) PTX3-3ʹUTR vector-transfected THP1-derived macrophages were treated with METTL3 or control siRNA (n = 4 cells).Renilla luciferase activity was measured and normalized to firefly luciferase activity.m Ptx3 mRNA stability in WT and Mettl3 KO BMDMs treated with actinomycin D at the indicated times point determined by RT-qPCR (n = 3 animals).Statistical analysis of the data was performed using two-sided unpaired t test with Welch's correction (j left) or not (d, h, j right, k), or 2-way ANOVA (i, l, m) followed by Sidak's multiple comparison tests.Data are presented as means ± SEM from one of three independent experiments.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s, not significant.promote mRNAs translation 16,19 , increasing evidence indicates that YTHDF3, through cooperation with YTHDF1 and YTHDF2, affects methylated mRNAs decay, by recruiting different complexes to regulate mRNAs clearance 37,38,53,54 .Here, we provide the evidence that the METTL3/YTHDF3 m 6 A axis preferentially affects the 3'UTR of PTX3 when triggering its decay during M2 macrophage activation.Mutations eliminating the RRACH motif can partially protect against this m 6 A-regulated degradation.The above findings suggest that the control of PTX3 mRNA decay by the METTL3/YTHDF3 m 6 A axis represents the mechanism for suppressing M2 macrophage activation.
Autophagy is an evolutionarily conserved process of recycling cytosol components and maintaining intracellular homeostasis.Accumulating evidence indicates that autophagy is essential for M2 macrophage activation.For example, impaired autophagy inside macrophages increased inflammation in obese mice by promoting M1 and decreasing M2 activation 41 ; CCL2 and IL-6 are potent inducers of autophagy in these cells, and they can trigger development along with the M2 phenotype 42 ; A knockdown of lincRNA-Cox2 in macrophages inhibited NLRP3 inflammasome activation and promoted autophagy, decreasing pro-inflammatory cytokine secretion 55 ; In the present study, KEGG pathway analysis revealed that molecules associated with autophagy were enriched in both PTX3 and METTL3-deficient macrophages.Moreover, METTL3/YTHDF3-m 6 A/PTX3 axis exerted its regulatory effects on autophagy maturation, which is a critical but often overlooked step of autophagy.A recent report showed that a set of SNARE complex proteins, including STX17, SNAP29, and VAMP8, are essential for autophagy maturation.The depletion of STX17 causes autophagosome accumulation and prevents their further degradation 40 ; The macrophage-specific V-ATPase subunit, ATP6V0D2, facilitates autophagy maturation and restricts macrophage inflammation through its interaction with STX17 39 .Here, we verified that the METTL3/YTHDF3-m 6 A/PTX3 axis played a vital role in the autophagy maturation of macrophage by regulating STX17 expression.Notably, through RNA-seq analysis, we also found that PTX3-deficient macrophages reduced the levels of key transcription factors such as ATF3 and MYC, and histone acetyltransferase KAT6A, which have been reported to modulate autophagy-associated gene expression 56,57 .Our observations demonstrate a strong link between autophagy and macrophage homeostasis mediated by the METTL3/ YTHDF3-m 6 A/PTX3 axis and its downstream targets.A detailed, comprehensive investigation into how the PTX3/STX17 axis modulates autophagy and macrophage homeostasis in allergic asthma is certainly needed.
A limitation of this study is that the detailed function of PTX3 has not been verified in Ptx3 conditional KO mice.In addition, although our studies with monocyte-derived macrophages in children with allergic asthma and myeloid cells in animal models have suggested a crucial role for METTL3/m 6 A modification in the development of allergic asthma mediated by macrophage activation, lung tissueresident macrophages evidence for this phenotype in childhood asthma is lacking, warranting further studies.Importantly, large cohorts of childhood allergic asthma need to be constructed, which evaluate the possibility of METTL3 or PTX3 levels as potential biomarkers for the diagnosis and assessment of childhood allergic asthma.
In summary, we provide compelling in vitro and in vivo evidence demonstrating the critical role of m 6 A modification in controlling macrophage homeostasis in allergic asthma.The combined network of 'writer' METTL3, 'reader' YTHDF3, and 'target' PTX3 molecules highlight an intriguing m 6 A-dependent gene regulatory mechanism.The insights gained in this study will help to further our understanding of the pathophysiological roles of m 6 A modification in macrophage homeostasis, and help develop therapeutic strategies for the treatment of allergic asthma.

Methods
Human study design 55 children with allergic asthma and 50 broadly age-and sex-matched healthy controls were recruited from the Children's Hospital of Fudan University.Informed consent was obtained from the parents of all participants.The patients with asthma were diagnosed according to the 2016 edition of the Guidelines for the Diagnosis and Prevention of Childhood Bronchial Asthma of China 5 .Individuals without a history of asthma and any other allergic diseases acted as controls.Detailed characteristics of study participants are presented in Supplementary Table 2.The study was approved by the Research Ethics Board of the Children's Hospital of Fudan University (No. 2020-81).
Human PBMCs were isolated using Ficoll-Paque density gradient solution (density=1.077g/ml, GE Healthcare).Plasma was isolated from peripheral blood by centrifugation at 400 g for 10 min.Peripheral blood was mixed with PBS and overlaid on top of Ficoll.Following centrifugation, PBMCs were aspirated from the Ficoll-plasma interface and washed twice in PBS.

Mice
Mettl3 flox/flox mice were generated by Shanghai Model Organisms Center, Inc (China), using a LoxP targeting system.Briefly, the Mettl3 targeting vector was designed to flank exon 2-3 with LoxP sites and electroporated into C57BL/6 embryonic stem cells (ES).Positive ES clones were confirmed by PCR and sequencing, and injected into C57BL/6 blastocysts to generate chimeric offspring.Chimeric mice were mated with C57BL/6 mice to obtain Mettl3 flox/flox mice.These Mettl3 flox/flox mice were crossed with Lyz2-Cre mice (The Jackson Laboratory) to generate Mettl3 fl/fl (WT) and Mettl3 fl/fl Lyz2 Cre/+ (KO) mice.Primers used to identify genetically modified mice are listed in Supplementary Table 3.All mice were housed, bred, and maintained under specific pathogen-free conditions, fed standard laboratory chow, and kept on a 12 h light/dark cycle and temperature and humidity were kept at 22 ± 1 °C, 55% ± 5%.Experiments complied with the relevant laws and institutional guidelines, as overseen by the Animal Studies Committee of the Children's Hospital of Fudan University.

Pulmonary function testing for assessing airway hyperresponsiveness (AHR)
We have previously described the assessment of AHR in mice with induced asthma in detail 13 .In brief, mice were anesthetized with phenobarbital sodium (50 mg/kg body weight), and PBS or increasing concentrations of aerosolized methacholine were nebulized into airways.Invasive measurements of AHR were made on a FinePointe Resistance and Compliance System (Buxco Research Systems).Airway resistance was calculated using FinePointe software.The normalization function of the software was used, and data were represented as a deviation from 0 mg/ml.

Depletion of macrophages
Macrophages were depleted using CLs (ClodronateLiposomes.org) as described previously 28 .Briefly, mice were anesthetized and i.p. injected with 200 µl clodronate liposomes every 72 h during CRE treatment.Controls included i.p injections of 200 μl PBS.For confirmation of depletion during allergic asthma, BALF was harvested and analyzed by flow cytometry for a reduction in SiglecF + Mac-3 + cells.

Immunohistochemistry and immunofluorescence
Sections (5 µm) were prepared from formalin-fixed paraffin-embedded (FFPE) lungs, and stained for hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS).An inflammation score on a scale of 0 to 4 was assigned to the perivascular and peribronchial areas of multiple fields per slide, based on previously published methodology 62 .A score of 0 represented a complete lack of inflammation, and a designation of 4 was assigned to areas where inflammatory cells were present at a depth of greater than 3 cells completely surrounding an airway or blood vessel.The numerical scores for the abundance of PAS-positive mucuscontaining cells in each airway were determined as follows: 0, <0.5% PAS-positive cells; 1, 5-25%; 2, 25-50%; 3, 50-75%; 4, >75%.For the detection of neutrophils, FFPE tissue slides were stained with anti-Gr1 (Cell Signaling Technology, #87048, 1:50).

Isolation of bone marrow-derived macrophages (BMDMs)
Mouse BMDMs were isolated and cultured as described previously 12 .Briefly, bone marrow was extracted from femurs and, after erythrocyte lysis, plated in complete medium, consisting of DMEM containing 10% FBS and 20 ng/ml macrophage colony-stimulating factor-1 (M-CSF, R&D System).Cells were allowed to differentiate for 7-8 days before use.

Isolation of alveolar macrophages
Isolation of mouse alveolar macrophages was performed according to our previously published work with some modifications 12,63 .Lung tissues harvested from WT and Mettl3 KO mice were minced and Fig. 6 | METTL3/YTHDF3 m 6 A axis degrades PTX3 mRNA during M2 macrophage activation.a Heatmap profiling downregulated genes in human METTL3deficient THP1-derived macrophages.b Western blot showing protein levels of YTHDF3 in both Mettl3 KO mice BMDMs (n = 3 animals) and METTL3-deficient THP1derived macrophages (n = 3 cells), respectively.c RT-qPCR analysis of the abundance of YTHDF3 in monocyte-derived macrophages from 55 childhood allergic asthma and 50 healthy controls.The detailed minimum, median, maximum, 25th, 75th percentile (box), and 5th and 95th percentile (whiskers) of box plots were provided in the Source data file.d RT-qPCR (left) analysis of M2-associated markers mRNA levels (n = 4 cells), and ELISA (right) detection of the IL-10 secretion levels (n = 4 cells for control group and n = 5 cells for knockdown groups) in THP1-derived macrophages transfected with YTHDF3 siRNAs pools (200 nM), followed by IL-4 stimulation.e Elevated levels of p-AKT and p-STAT6 in YTHDF3-deficient THP1derived macrophages were detected by Western blot (n = 3 cells).f RT-qPCR (n = 3 cells) and ELISA (n = 3, 6, 4 cells for Ctrl LV+siNC, METTL3 LV+siNC, METTL3 LV +siYTHDF3 group, respectively) showing M2 activation-associated markers expression in THP1-derived macrophages overexpressing METTL3, with or without YTHDF3 knockdown.g YTHDF3 RIP-analysis of PTX3 m 6 A level in control or METTL3deficient THP1-derived macrophages (n = 7 cells for control group and n = 8 cells for knockdown group).Relative enrichment was normalized by input.h The increase of PTX3 mRNA (n = 3 cells) and secretion levels (n = 3 cells for control group and n = 5 cells for knockdown group) in YTHDF3-deficient THP1-derived macrophages was quantitated by qPCR and ELISA.i Western blot showing up-regulated PTX3 protein levels in YTHDF3-deficient THP1-derived macrophages (n = 3 cells).j Effect of YTHDF3 knockdown on PTX3 mRNA stability in THP1-derived macrophages treated with actinomycin D (n = 4 cells).k Dual-luciferase reporter assays demonstrating the effect of YTHDF3 on PTX3 3ʹUTR reporters with either WT or mutated m 6 A site (n = 4 cells).Statistical analysis of the data was performed using two-sided unpaired t test (b, d, h, i), Mann-Whitney test (c), 1-way ANOVA (f right), and 2-way ANOVA (e, f left, g, j, k) followed by either Tukey's or Sidak's multiple comparison tests.Data are presented as means ± SEM from one of three independent experiments.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s, not significant.collected into gentleMACS C Tubes, containing the enzyme mix of the Lung Dissociation Kit (Miltenyi Biotech).Tissues in C Tubes were subjected to the run program 37 °C_m_lung for 30 min on the gentle-MACS Octo Dissociator.Cell suspensions were passed through a 70 µm MACS SmartStrainer (Miltenyi Biotech), followed by centrifugation at 300 g for 7 min at 4 °C.All isolated cells were further purified by EasySep Mouse F4/80 Positive Selection Kit (STEMCELL) and macrophage adherence, and stimulated by IL-4 for 12 h.The percentage of alveolar macrophages in purified cell populations was detected by flow cytometry analysis using anti-CD11c and F4/80 antibodies.

Cell culture, differentiation, and transfection
Human THP1 cells (obtained from ATCC) were maintained in RPMI-1640 medium containing 10% heat-inactivated FBS.To generate macrophage-like cells, the seeded THP1 cells were treated with 200 nM PMA (Sigma-Aldrich) for 48 h.For M1 and M2 activation, macrophages were stimulated with LPS (200 ng/ml, 3 h, Sigma-Aldrich) or IL-4 (20 ng/ml, 24 h, R&D Systems), respectively.Small interfering RNAs (siRNAs), including PTX3 shRNA lentivirus (PTX3 shRNA), and METTL3overexpressing lentivirus (METTL3 LV) were synthesized by Gene-Pharma (Shanghai, China).The sequences of siRNAs and controls are provided in Supplementary Table 4. THP1 cells were transfected with these RNAs using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer's instructions.m 6 A-seq assay and data analysis METTL3-knockdown human THP1-derived macrophages were incubated with IL-4 for 24 h.Total RNA was isolated and mRNAs with polyA were enriched using Oligo-dT.RNA fragmentation reagent (10 mM ZnCl 2 , 10 mM Tris-HCl pH 7.0) was used to randomly fragment RNA, and an m 6 A antibody (Synaptic Systems) was applied for m 6 A pulldown (m 6 A-IP).Both input and m 6 A IP samples were prepared for nextgeneration sequencing.The library preparation was constructed using the TruSeq Stranded mRNAs Sample Prep Kit (Illumina), and then was deeply sequenced on the Illumina Hiseq 4000 at OE Biotech.Co. Ltd (Shanghai, China).
For data analysis, the reads from input and m 6 A IP samples were aligned to the human genome GRCh38 using HISAT2 with default parameters.The differentially expressed transcripts of m 6 A methylome between METTL3-knockdown and Ctrl macrophages were detected by MeTDiffpeak calling software.Sequence motifs enriched in m 6 A peak regions compared to control regions were identified using MEME.Integrative Genomics Viewer (IGV) was used to visualize the distribution of the m 6 A peaks.

RNA-seq assay and data analysis
Total RNA was isolated from METTL3 or PTX3-knockdown THP1derived macrophages stimulated by IL-4 for 24 h.The TruSeq RNA Sample Preparation Kit (Illumina) was used for library preparation.Libraries at a final concentration of 10 pM were clustered onto a single read flow cell by cBot and sequenced to 150 bp using an Illumina NovaSeq 6000 instrument (Illumina).Library construction and sequencing were performed by Sinotech Genomics.Co. Ltd (Shanghai, China).Differential genes expression were analyzed by the standard Illumina sequence analysis pipeline.To comprehensively analyze the RNA-seq data, filtering criteria were applied (≥1.5-fold change, P < 0.05).

Quantitative real-time PCR
Total RNA was extracted from cells using Trizol Reagent.cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara).To quantify mRNA expression, cDNAs were amplified by RT-qPCR using the SYBR Premix Ex Taq RT-PCR kit (Takara).ACTIN levels were used as internal housekeeping controls in both human and mouse samples.The data was collected using Roche LC 480.Fold-change in expression was calculated using the 2 -ΔΔCt method.The primer sequences for all genes are provided in Supplementary Table 3.

Enzyme-linked immunosorbent assay (ELISA)
The protein levels of IL-10, TNF, IL-4, IL-13, and PTX3 were measured in culture medium, serum, and plasma with the DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions.

The SELECT detection assay
The SELECT was performed using the Epi-SELECT m 6 A site identification Kit (Epbiotek, Guangzhou, China) as described previously 64 .In brief, 1 μg total RNA was mixed with 1 μM Up Primer, 1 μM Down Primer, and 2 μl dNTP in 2 μl 10× Reaction buffer.Annealing of RNA and primers was performed for 1 min at each 90 °C, 80 °C, 70 °C, 60 °C, 50 °C, and 6 min at 40 °C.Subsequently, the former mixture, DNA polymerase, ligase, and ATP were added in a total volume of 20 μl and incubated for 20 min at 40 °C and 80 °C.The qPCR reaction was performed using SYBR Premix.Relative SELECT products were calculated by normalization to the RNA abundance determined by the control.The primer sequences are provided in Supplementary Table 3.

MeRIP-qPCR
The Magna MeRIP m 6 A Kit (Millipore) was used to perform MeRIP-qPCR according to the manufacturer's instructions.200 µg of total RNA was extracted from THP1-derived macrophages infected with METTL3 siRNA or control siRNA.RNA was sheared to approximately 100 nt in length and incubated with m 6 A antibody (Millipore, MABE1006, 2 μg/ml)-or IgG-conjugated Protein A/G Magnetic beads at 4 °C overnight.Methylated RNAs were immunoprecipitated with beads, eluted, and recovered with the RNeasy kit (QIAGEN).One-tenth of fragmented RNA was saved as input control and further analyzed by RT-qPCR along with the MeRIPed RNAs using primers listed in Supplementary Table 3.The related enrichment of m 6 A in each sample was h Heatmap identifying the down-regulated transcripts in PTX3-deficient THP1-derived macrophages.i RT-qPCR (n = 4 cells) and immunoblot (n = 3 cells) verifying reduced levels of STX17 in PTX3-knockdown BMDMs.j RT-qPCR showing up-regulated STX17 expression in Mettl3 KO BMDMs (n = 4 animals for WT group and n = 3 animals for KO group) and YTHDF3-deficient THP1-derived macrophages (n = 5 cells for control group and n = 4 cells for knockdown group), respectively.k The increased STX17 levels in Mettl3 KO BMDMs (n = 3 animals) and YTHDF3-deficient THP1-derived macrophages (n = 3 cells) by Western blot.l Immunoblot analysis of LC3, STX17, and PTX3 levels in control and PTX3-knockdown BMDMs treated with rapamycin for 0-6 h (n = 3 cells).Statistical analysis of the data was performed using two-sided unpaired t test with Welch's correction (i left) or not (a, b, d, e, g, i right, j, k), and 2-way ANOVA (l) followed by Sidak's multiple comparison tests.Data are presented as means ± SEM from one of three independent experiments.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4 |
Fig.4| METTL3 regulates PTX3 mRNA stability and expression through manipulation of m6 A modification.a The predominant consensus m6 A motif RRACH was detected in both control and METTL3-deficient macrophages through m 6 A-seq.b Density distribution of m 6 A peaks across mRNA transcripts.c The proportion of m 6 A peaks distribution in the 5ʹUTR, CDS, and 3ʹUTR regions across mRNA transcripts in control and METTL3-deficient macrophages.d Distribution of peaks (>1.2-fold change, P < 0.05) with a significant change in both mRNAs expression and m6 A level in METTL3-deficient macrophages relative to control cells.e Venn diagram showing the number of overlapping genes between m 6 A-Hypoupregulated genes in METTL3-deficient macrophages and M2 activation-associated functional genes.f Spearman correlation (two-sided) between METTL3 and PTX3 mRNA levels in monocyte-derived macrophages from 55 childhood allergic asthma populations.g Integrative Genomics Viewer (IGV) showing the m 6 A abundance on PTX3 mRNA transcripts in METTL3 deficiency and control macrophages as detected by m 6 A-seq.h SELECT assay showing the relative amount of qPCR products targeting the AGA 1333 CT site on PTX3 3ʹUTR in THP1-derived macrophages transfected Article https://doi.org/10.1038/s41467-023-43219-wNature Communications | (2023) 14:7328

Fig. 8 |
Fig. 8 | Enhanced levels of PTX3 are associated with disease severity of childhood allergic asthma.The PTX3 mRNA levels in monocyte-derived macrophages (a) and protein levels in the plasma (b) were measured in 55 childhood allergic asthma and 50 healthy controls using RT-qPCR and ELISA, respectively.c RT-qPCR showing elevated levels of STX17 in monocyte-derived macrophages from 55 childhood asthma population.The detailed minimum, median, maximum, 25th, 75th percentile (box), and 5th and 95th percentile (whiskers) of box plots were provided in the Source data file.d ROC curve analysis of PTX3 protein levels in 55 childhood asthma.e Spearman correlation (two-sided) analysis between PTX3 protein levels in plasma and blood eosinophils numbers, or FeNO in 55 childhood allergic asthma.f Proposed working model of m 6 A-controlled macrophage homeostasis in allergic asthma (Schematic created with BioRender.com).Statistical analysis of the data was performed using two-sided unpaired t test (b), or Mann-Whitney test (a, c).Data are presented as means ± SEM. **P < 0.01, ****P < 0.0001.