Response to Ribeiro Jr. et al.

We appreciate Ribeiro Junior et al. [1] for their interest and thoughtful comments regarding our recent correspondence [2]. We appreciate the opportunity to further address these remarks, beyond what was already discussed in the limitations of our study. We certainly agree that socioeconomic status (SES) and its associated risk factors (the “exposome”) may play a role in creating a baseline systemic inflammatory state in different individuals. Furthermore, it is certainly possible that residents in our control region (Scarsdale) have more access to protections from outdoor pollution secondary to their SES. Though an attractive idea, there is unfortunately no comprehensive measurement at this time that can adequately quantify the “exposome,” thus limiting many epidemiologic studies in their ability to measure all that an individual is susceptible to. For this reason, it was unable to be controlled for, as mentioned in our limitations. Creating a measure such as this would be an admirable endeavor and would certainly further contribute to studies like ours. Similarly, we do agree that occupational exposures, like benzene, are established risk factors for MDS and thus have noted them as potential confounders. However, the purpose of this study was to evaluate hazardous air pollution, not known mutagenic occupational hazards that have been previously studied. We estimate occupational exposures affecting only a minor percentage of our cohort. Migration throughout our 17-year study period was also noted as a limitation in our study. However, our approach nonetheless focused on long-term ambient exposure over this period, rather than the episodic, high-intensity exposures seen with intercontinental wildfire smoke. These exposures tend to have intense particulate loads that may induce different mutagenic pathways than chronic air pollution. Our previous work showcased that World Trade Center-exposed first responders experienced high burden clonal hematopoiesis and may better mimic acute exposure like wildfire smoke [3]. Nonetheless, we do view acute exposures such as this to be complementary to the chronic insult of air pollution. Regarding the concerns raised about our murine model, we emphasize that while intratracheal (IT) instillation is not as physiological as natural inhalation, it is a methodologically necessary approach in laboratory settings. This technique guarantees the delivery of a standardized dose of our PM2.5 cocktail, which was specifically formulated to mimic urban industrial and traffic pollutants, directly into the lung. By introducing the pollutants precisely into the respiratory tract, our IT technique effectively replicates the physical presence of particulate matter observed in human lung tissue and airway macrophages [4, 5]. These engulfed particles are known to trigger persistent cytokine secretion, particularly IL-6, TNF-a, IL-1β, promoting both localized and systemic inflammatory responses [6]. Our model reflects this process, allowing for the investigation of downstream hematopoietic events within a controlled experimental timeframe. The validity of using IT delivery for particulate matter in murine models is further established in the field, most notably by Hill et al., who utilized this method to study the promotion of lung cancer by particulate matter [7]. To specifically refute the suggestion that the observed defects might be procedural artifacts, we point to our clonogenic assays where human CD34+ hematopoietic cells were treated directly with BC in vitro. These experiments resulted in impaired growth, confirming that the hematopoietic defects are a direct consequence of BC exposure rather than the administration process itself. Ultimately, we agree that more granularity is required when studying air pollution exposure in humans. Doing so is necessary to guide policy aimed at mitigating climate- and pollution-related health risks. We invite Ribeiro Junior et al. and others to continue dialogue and collaborate with us in exploring this further and identifying solutions. Sincerely, Matthew P. Davidsohn, Divij Verma, Yiyu Zou, Amit Verma, Aditi Shastri. The authors have nothing to report. Amit Verma: Stelexis, Roshon and ClinStreet: Equity holder; Stelexis: Honoraria; Halia: Research Funding; Curis: Research Funding; Medpacto: Research Funding; BMS: Research Funding; Prelude: Research Funding. Aditi Shastri: NACE & PeerView: Honoraria; Jassen: Consultancy; Gilead, Rigel, Kymera: Consultancy; Kymera: Research Funding; Ryvu therapeutics: Research Funding; Geron: Honoraria. The other authors declare no conflicts of interest. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.