Smoke lowers RNA methylation enzyme WTAP, stabilizing pIgR to curb lung inflammation in mice

Smoke inhalation injury is among the leading causes of death in fire-related incidents, and it frequently occurs alongside severe burn trauma. While heat from inhaled smoke mainly damages the upper airway, the smoke itself carries carbon monoxide, cyanide, and abundant toxic particles smaller than one micrometer in diameter, which can travel deep into the lower respiratory tract. There, these components injure alveolar epithelial and vascular endothelial cells, break down the alveolar-capillary barrier, and raise microvascular permeability, allowing protein-rich fluid to flood the alveolar spaces. The damaged epithelial barrier triggers a rapid innate immune response, recruiting alveolar macrophages and neutrophils and unleashing a cytokine storm dominated by TNF-α, IL-1β, and IL-6. If this cascade amplifies unchecked, it can progress to acute respiratory distress syndrome, irreversible respiratory failure, and multiple organ dysfunction. Despite advances in airway management, fluid resuscitation, and supportive care, the specific mechanisms governing the epithelial response to smoke inhalation have remained poorly understood.

A new study now points to a post-transcriptional regulatory pathway that appears to act as an endogenous brake on this injury process. The researchers report that smoke exposure reduces global levels of N6-methyladenosine, or m6A, the most abundant internal chemical modification found on eukaryotic messenger RNA, and that the methyltransferase WTAP, a key component of the core m6A-writing complex, is significantly downregulated after injury. When the team silenced WTAP in lung epithelial cells, smoke-induced inflammation and apoptosis were further attenuated rather than worsened, suggesting that the natural decline in WTAP during smoke exposure may represent a protective, self-limiting response. Through this drop in WTAP activity, the cells reduce m6A methylation on the messenger RNA encoding the polymeric immunoglobulin receptor, or pIgR, which stabilizes that transcript, raises pIgR expression, and helps limit early pulmonary inflammation and epithelial cell death.

To reach these conclusions, the investigators first built a smoke generator consisting of a burning chamber and an animal chamber, burning pine sawdust as a cost-effective fuel representative of common indoor fire materials such as wood, paper, and plastic. Real-time monitoring of the animal chamber showed that oxygen concentrations fell over the first five minutes of exposure while carbon dioxide, carbon monoxide, and sulfur dioxide rose in a time-dependent manner, replicating the hypoxic and toxic environment of a real fire. In mice subjected to this exposure, the alveolar-capillary barrier was clearly disrupted: total protein concentrations in bronchoalveolar lavage fluid rose at 24 and 48 hours after injury, and lung tissue sections revealed massive inflammatory cell infiltration, erythrocyte exudation, and protein debris deposition within the alveolar spaces. The tracheal wall structure remained relatively intact, a pattern the authors attribute to the acute nature of the injury, since airway wall thickening is more typical of chronic remodeling.

Accompanying this structural damage, the expression of the pro-inflammatory cytokines TNF-α and IL-1β surged in lung tissue, while the anti-apoptotic protein Bcl-2 declined significantly according to RT-qPCR analysis. Western blotting also documented activation of STAT3, a key transcriptional mediator of the acute-phase response and of innate immunity and cytokine signaling. Parallel experiments in MLE-12 mouse lung epithelial cells treated with smoke extract mirrored these findings: cell viability and migratory capacity, measured by CCK-8 and scratch assays, declined in a dose- and time-dependent manner, TNF-α and IL-1β rose, Bcl-2 fell, and flow cytometry confirmed a significant increase in the rate of epithelial apoptosis. Together, these data established that smoke exposure drives severe epithelial injury through combined inflammatory and apoptotic signaling in both the intact animal and cultured cells.

The search for post-transcriptional changes began with colorimetric quantification of overall m6A methylation, which revealed a significant reduction both in the injured mouse lungs and in smoke-exposed epithelial cells. This decrease is consistent with earlier reports that global m6A levels fall in pulmonary epithelial cells exposed to environmental particulates, and that m6A methylation declines in allergic asthma models; it also matches observations of reduced m6A levels in the peripheral blood of chronic smokers. To pinpoint the enzymes responsible, the team measured the messenger RNA levels of the major m6A writers, erasers, and readers, including the methyltransferases METTL3, METTL14, and WTAP, the demethylases ALKBH5 and FTO, and the reader YTHDF2. Among all of these, WTAP showed the most significant downregulation in vivo and in vitro, identifying it as the likely primary mediator of the observed loss of global m6A methylation after smoke exposure.

Because WTAP expression declined naturally after smoke exposure, the researchers hypothesized that this drop might be an endogenous protective mechanism rather than a purely harmful consequence of injury. They generated a stable WTAP-knockdown MLE-12 cell line using lentiviral vectors and exposed it to smoke extract. Compared with control cells, the WTAP-silenced cells mounted a markedly weaker inflammatory response: TNF-α and IL-1β expression fell substantially, Bcl-2 levels rose, and flow cytometry showed a lower apoptosis rate. Western blot analysis further revealed that both total STAT3 and phosphorylated STAT3 were reduced in the WTAP-knockdown cells after smoke exposure, indicating that WTAP may contribute to inflammatory signaling in part through modulating the STAT3 pathway. The authors note that this finding aligns with prior work showing that depletion of the m6A writer METTL14 inhibits viral replication, that WTAP depletion limits overactive interferon signaling by reducing m6A methylation of IRF3 and IFNAR1 messenger RNA, and that WTAP can behave as a pro-inflammatory amplifier in other contexts, such as ulcerative colitis, pulmonary fibrosis, and LPS-induced sepsis, underscoring that WTAP’s role is highly context-specific.

To identify the direct downstream target through which WTAP exerts these effects, the team re-analyzed transcriptomic data from the lungs of smoke-injured mice, applying criteria of p less than 0.05 and an absolute log2 fold change greater than 1, which yielded 496 upregulated and 180 downregulated genes. Weighted gene co-expression network analysis then identified a black module showing the strongest positive correlation with the smoke inhalation injury phenotype, with a correlation coefficient of 0.96 and p less than 0.01, and a dark turquoise module showing the most significant negative correlation at minus 0.99 with p less than 0.001. Overlap analysis produced 235 genes shared between the black module and the upregulated differentially expressed genes, and 46 genes shared between the dark turquoise module and the downregulated genes. Among these candidates, the polymeric immunoglobulin receptor showed the most significant upregulation after smoke exposure. The m6A2Target database independently supported pIgR as a highly conserved direct target of WTAP-mediated m6A methylation across different tissues and species.

Functional and mechanistic experiments then connected pIgR to the protective effect of WTAP loss. The SRAMP prediction database identified very high confidence m6A binding sites at positions 756 and 772 of the pIgR transcript, and high confidence sites at positions 1356 and 3750. MeRIP-qPCR confirmed strong m6A enrichment within the 756/772 region of pIgR messenger RNA, whereas the 1356 and 3750 sites showed no significant enrichment and were therefore not functional modification sites in this model. When WTAP was silenced, m6A methylation within the 756/772 region dropped significantly after smoke exposure, and actinomycin D chase experiments showed that the half-life of pIgR messenger RNA was prolonged, indicating that WTAP-mediated methylation normally promotes pIgR transcript decay. For context, smoke extract alone extended the pIgR messenger RNA half-life from 1.645 hours in control cells to 2.557 hours. Silencing pIgR alone in MLE-12 cells intensified inflammation, raising TNF-α and IL-1β while lowering Bcl-2, and in double-knockdown rescue experiments, pIgR silencing reversed the anti-inflammatory and anti-apoptotic effects of WTAP knockdown at both the messenger RNA and protein levels.

The biological plausibility of pIgR as a protective factor rests on its established roles in mucosal immunity. pIgR is a transmembrane protein on mucosal epithelial cells that transports dimeric IgA across the epithelium to form secretory IgA on the mucosal surface, mediating immune exclusion and preventing pathogen adhesion throughout the respiratory and gastrointestinal tracts. Beyond transport, pIgR is considered important for maintaining immune homeostasis: pIgR-deficient mice show enhanced airway neutrophil infiltration and disrupted mucosal immune balance, and loss of pIgR in mice produces a COPD-like phenotype coupled to bacterial invasion, neutrophilic infiltration, and progressive small-airway remodeling. The finding that pIgR rises when WTAP falls therefore fits a broader picture in which preserving pIgR expression protects against airway inflammatory injury. The authors caution, however, that although WTAP knockdown suppressed STAT3 activation and pIgR knockdown reversed the protective effects, these observations came from separate experimental arms and represent an association rather than a directly established causal link between pIgR and STAT3; direct validation through a pIgR cytoplasmic-tail truncation mutant or co-immunoprecipitation is planned.

Several limitations frame the scope of these results. m6A modification is a dynamic process involving multiple writers, erasers, and readers, and although WTAP emerged as the principal player here, other methyltransferases may contribute; whether specific reader proteins mediate the effect of WTAP on pIgR messenger RNA stability also remains to be determined. As a transmembrane protein, pIgR’s function is spatially restricted, and this study examined only its association with inflammation, leaving its membrane localization on alveolar epithelial cells and its interaction with secretory IgA for future work. The team also did not perform an in vivo AAV-mediated knockdown or rescue experiment, so the axis has so far been established mainly in cells, and the MeRIP-qPCR primers spanned the adjacent 756 and 772 sites together, so single-site contributions could not be distinguished without site-directed mutagenesis. Even with these caveats, the study highlights post-transcriptional RNA regulation as a previously underappreciated layer of the epithelial response to smoke inhalation, and it proposes the WTAP/m6A/pIgR axis as an endogenous protective pathway and a potential target for early intervention in smoke inhalation injury.

Subject of Research: WTAP-mediated m6A methylation of pIgR mRNA in the epithelial response to smoke inhalation injury

Article Title: WTAP-mediated m6A methylation stabilizes pIgR mRNA and attenuates smoke-induced epithelial inflammation and apoptosis

Article References: Li, J., Huang, S., & Guo, G. (2026). WTAP-mediated m6A methylation stabilizes pIgR mRNA and attenuates smoke-induced epithelial inflammation and apoptosis. iScience, 29(11), Article 117958. https://doi.org/10.1016/j.isci.2026.117958

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117958

Keywords: smoke inhalation injury, m6A methylation, WTAP, pIgR, mRNA stability, STAT3, epithelial apoptosis, pulmonary inflammation, lung epithelial cells, secretory IgA, post-transcriptional regulation, acute lung injury