FMRP, an RNA-binding protein induced by the Mycoplasma pneumonia CARDS toxin, regulates multiciliogenesis and inflammation

To the Editor: Mycoplasma pneumoniae (M. pneumoniae), an atypical pathogen lacking a cell wall, is a leading cause of community-acquired pneumonia in children; it primarily affects school-age children.[1] The community-acquired respiratory distress syndrome (CARDS) toxin, an adenosine diphosphate-ribosylating and vacuolating exotoxin unique to M. pneumoniae, induces cell swelling and vacuolization.[2] Thus far, the precise functional characteristics of the CARDS toxin have not been fully elucidated. Motile cilia, which extend from specialized epithelial surfaces, perform essential roles during development and disease by facilitating the movement or clearance of fluids and particles through coordinated beating.[3] Fragile X mental retardation protein (FMRP), encoded by the Fmr1 gene, is widely expressed in organs throughout the body. FMRP is enriched in the axonemal central lumen; it has critical roles in multiciliated cell differentiation and multiciliogenesis.[4] A recent report indicated that FMRP is also expressed in the lungs, where it protects the airways from xenobiotic stress.[5] In this study, we investigated whether ciliary FMRP plays a role in the pulmonary immune response during M. pneumoniae infection. All animal experiments were approved by the Capital Medical University Animal Care and Use Committee (No. AEEI-2024-117). BALB/c mice were intratracheally instilled with recombinant CARDS toxin (700 pmol/L) to simulate M. pneumoniae infection, and heat-inactivated CARDS toxin (15 min at 100°C) was used as a control. Seven days after the procedure, tracheal tissue, lung tissue, and bronchoalveolar lavage fluid (BALF) samples were collected and analyzed [Supplementary Material and Supplementary Figure 1A, https://links.lww.com/CM9/C306]. Compared with control mice, CARDS-treated mice exhibited minor body weight loss [Supplementary Figure 1B, https://links.lww.com/CM9/C306]. Hematoxylin–eosin staining revealed bronchial epithelial damage, thickened alveolar septa, and inflammatory cell infiltration in the lungs of CARDS-treated mice [Figure 1A]. Additionally, BALF protein levels, proinflammatory cytokine expression, and immune cell infiltration were increased in the lungs of CARDS-treated mice [Supplementary Figure 1C–F, https://links.lww.com/CM9/C306]. These results suggested that recombinant CARDS toxin induces inflammatory responses in mice.Figure 1: FMRP induced by CARDS toxin regulates multiciliogenesis and inflammation. (A) Representative image of hematoxylin-eosin staining of lung sections. Red arrows indicate bronchial epithelial damage, blue arrows indicate alveolar septum thickening, and black arrows indicate the infiltration of inflammatory cells. Insets show enlarged views of the boxed regions. Scale bars = 2 mm (main image) and 100 μm (magnified region). (B) Representative image of whole-mount immunofluorescence staining of trachea tissue. Tracheas were stained with anti-β Ⅳ tubulin antibody (green) and Hoechst (blue). Insets show enlarged views of the boxed regions in the trachea. Scale bars = 20 μm (main image) and 10 μm (magnified region). (C) Immunoblotting shows the protein level of FMRP in the lung of control and CARDS treated mice. β-actin was used as a loading control. (D) Left, quantitative real-time polymerase chain reaction analysis of Fmr1 expression. Right, immunoblotting shows the expression of FMRP. (E) Quantitative real-time PCR analysis of proinflammatory cytokines in the lung. All data are presented as mean ± standard error of mean. Statistics indicate significance by one-way analysis of variance with Bonferroni correction (D) and unpaired t-test (E). * P <0.01; † P <0.001; CARDS: Community-acquired respiratory distress syndrome; CCL: C-C motif chemokine ligand; DHPG: Dihydroxyphenylglycine; FMRP: Fragile X mental retardation protein; IL: Interleukin; ns: Not significant; PBS: Phosphate buffered saline; PCR: Polymerase chain reaction; TNF-α:Tumor necrosis factor alpha.RNA-seqiencing (RNA-seq) analysis was performed to compare transcriptome signatures between control and CARDS-treated mice. Principal component analysis (PCA) revealed clear separation in transcriptome profiles between the control and CARDS-treated groups [Supplementary Figure 2A, B, https://links.lww.com/CM9/C306]. Gene set enrichment analysis and Gene Ontology annotation indicated that the differentially expressed genes were highly enriched in pathways related to motile cilia, cilium organization, and cilium movement. These genes are required for normal ciliary structure and function in mammals, including processes such as axial elongation, microtubule assembly, ciliary trafficking, and formation of the transition zone and basal body [Supplementary Figure 2C–E, https://links.lww.com/CM9/C306]. These findings demonstrated that the CARDS toxin disrupts normal ciliary gene expression. To validate the ciliary damage revealed by transcriptomic analysis, we examined the distribution and structure of cilia in mouse tracheal tissue using hematoxylin–eosin staining and whole-mount immunofluorescence. The results showed that motile cilia in tracheal tissue from CARDS-treated mice exhibited defects [Figure 1B, Supplementary Figure 3A, https://links.lww.com/CM9/C306]. Additionally, the number of basal cells (krt5-positive cells) increased in tracheal tissue from CARDS-treated mice, indicating damage to the epithelial barrier; these basal cells were primed for proliferation and differentiation [Supplementary Figure 3B, https://links.lww.com/CM9/C306]. The expression levels of cilia-related genes also were decreased in the lungs of CARDS-treated mice [Supplementary Figure 3C, https://links.lww.com/CM9/C306]. As previously mentioned, the Fmr1 gene encodes FMRP, which is enriched in the axonemal central lumen and participates in both multiciliated cell differentiation and multiciliogenesis.[4] Compared with the control group, CARDS-treated mice exhibited downregulation of FMRP expression; this reduction was accompanied by ciliary injury [Figure 1C, Supplementary Figure 3D, https://links.lww.com/CM9/C306]. BALF samples were collected from patients with M. pneumoniae pneumonia (MPP) and patients with non-MPP diseases (i.e., pneumonia caused by other pathogens). The clinical portion of this study was approved by the Ethics Committee of Beijing Chaoyang Hospital (2021-Ke-501). Written informed consent was provided. Proteomic analysis of the BALF samples was performed using liquid chromatography–tandem mass spectrometry [Supplementary Figure 4A, https://links.lww.com/CM9/C306]. PCA revealed clear differences between the MPP and non-MPP groups [Supplementary Figure 4B, https://links.lww.com/CM9/C306]. Gene set enrichment analysis and Gene Ontology analysis indicated that proteins related to cilium morphogenesis, movement, and organization were downregulated in MPP, consistent with the transcriptomic data from experimental mice [Supplementary Figure 4C, D, https://links.lww.com/CM9/C306]. FMRP expression also was downregulated in BALF samples from patients with MPP [Supplementary Figure 5A, B, https://links.lww.com/CM9/C306]. Collectively, these results suggested that FMRP expression is affected by M. pneumoniae infection. Dihydroxyphenylglycine (DHPG), an agonist of group I metabotropic glutamate receptors, can upregulate FMRP expression in neurons.[6] After applying DHPG to mouse tracheobronchial epithelial cells, we observed significant increases in Fmr1 expression (mRNA level) and FMRP expression (protein level) [Figure 1D]. In mice that received DHPG treatment 5 days after injection of CARDS toxin, the toxin-mediated changes in FMRP expression and ciliary morphology were reversed. Furthermore, lung tissue damage, cytokine gene expression levels, and the percentages of CD68+ alveolar macrophages and Ly6G+ neutrophils were significantly reduced after DHPG treatment [Figure 1E, Supplementary Figure 6, https://links.lww.com/CM9/C306]. Taken together, these findings imply that pharmacological activation of FMRP can protect the lungs from CARDS toxin-induced damage. In conclusion, our study demonstrates that CARDS toxin damages motile cilia in the respiratory tract and decreases FMRP expression in ciliated cells. These changes are consistent with observations in patients with MPP. FMRP might play a key role in CARDS-mediated ciliary injury and the maintenance of pulmonary immune homeostasis. Approaches that target the cilia–FMRP axis may help to address drug resistance in the treatment of M. pneumoniae infection. Funding This work was supported by Beijing Chao-Yang Hospital Golden Seeds Fundation (No. CYJZ202312), the Capital's Funds for Health Improvement and Research (No. 2022-1-1061) and Beijing Research Ward Demonstration Construction Project (No. BCRW202110). Conflicts of interest None.