Background: Methyltransferase 3 is a component with methyltransferase activity in the N6-adenosine methyltransferase complex. It can transfer the methyl group from S-adenosylmethionine (SAM) to adenosine (A) in mRNA, methylating it into N6-methyladenosine (m6A). Abnormal expression of the METTL3 gene plays an important regulatory role in the cell development and differentiation in various cancers and cardiovascular diseases. However, its involvement in regulating macrophages in interstitial lung diseases remains unexplored. This study aims to investigate the role of METTL3 in the activation of profibrotic macrophages in pulmonary fibrosis.

Objectives: The objective of this study is to elucidate the role of METTL3 in the activation of profibrotic Spp1 ⁺ macrophages and in pulmonary fibrosis. We aim to assess METTL3 expression in lung fibrotic tissues and in experimental mouse models, and to evaluate the impact of METTL3 modulation on the function of profibrotic Spp1 ⁺ macrophages. Furthermore, we seek to identify the molecular mechanisms of the downstream gene network regulated by METTL3 through m6A methylation in the fibrotic niche of in vivo lung tissue, thereby revealing the biological functions and roles of m6A methylation modification in the occurrence and development of pulmonary fibrosis.

Methods: The scRNA-seq data from lung tissue of IPF patients and healthy controls revealed that during the early development of IPF, the population of SPP1 ⁺ macrophages significantly increased, and these cells exhibited pro-fibrotic functions. The scRNA-seq and scATAC-seq data from bleomycin-induced pulmonary fibrosis showed the same results. Moreover, the m6A RNA methyltransferase METTL3 was primarily expressed in the SPP1 ⁺ macrophage population in the single-cell sequencing of lung tissues from IPF patients and mouse models. METTL3 expression in SPP1 ⁺ macrophages was analyzed in idiopathic pulmonary fibrosis (IPF) patients and pulmonary fibrosis models using immunofluorescence. METTL3 expression was modulated via knockdown/overexpression and knockout techniques both in vitro and in vivo. The pharmaceutical potential of targeting METTL3 was explored using the selective small molecule inhibitor STM2457 in bleomycin-induced pulmonary fibrosis. MeRIP-Seq was used to identify METTL3 target genes in macrophages.

Results: The scRNA-seq data of lung tissue from IPF patients and healthy controls showed that SPP1 ⁺ macrophages population significantly increased in fibrotic lung, and these cells exhibited pro-fibrotic functions. The same cell population was found in scRNA-seq data of experimental mouse models. Moreover, the METTL3 was mainly expressed in the SPP1 ⁺ macrophage population in the single-cell sequencing of lung tissues from IPF patients. METTL3 expression was downregulated in SPP1 ⁺ macrophage from fibrotic lungs compared to non-fibrotic controls. Decreased METTL3 expression was also observed in murine pulmonary fibrosis model. METTL3 knockdown in murine myeloid cell aggravated both IL4-induced bone marrow derived macrophage and peripheral blood monocytes derived macrophage activation. The supernatant of the METTL3 knockdown macrophages induced the fibroblast to myofibroblast transition with high expression of collagen. Myeloid cell specific knockout of METTL3 exacerbated bleomycin-induced pulmonary fibrosis. Inhibitor STM2457 treatment also exhibited enhance fibrotic effects in murine model. meRNA-Seq and RNAseq studies identified MafB is upregulated in METTL3 knockout marcrophages as an upstream regulator of Spp1. Mechanistically, METTL3 was found to modulate MafB, a key transcription factor in macrophage fate and activation. METLL3 regulated MafB in m6A dependent manner. Western blots confirmed that siRNA knockdown of MafB diminished the upregulation of profibrotic proteins IRF4 and Arg1.

Conclusion: Our research identifies METTL3 as a key modulator of SPP1 ⁺ macrophage activation during the remodeling of lung fibrotic tissue through regulation of MafB and Irf4 transcription factors. Our findings broaden the understanding and recognition of idiopathic pulmonary fibrosis (IPF).

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.