D17-04 Unique Role of Il-10 in Immune Priming Lung Immune Tone Induced by Mixed Oral Commensals Against Streptococcus Pneumoniae Infection

Abstract Rationale The lung microbiota plays an essential role in shaping local immunity. We showed lower airway microbial alterations caused by intratracheal inoculation of mixed oral commensals (MOC: Streptococcus mitis, Prevotella melaninogenica, Veillonella parvula) induced priming and recruitment of immune cells, thereby enhancing host defense against Streptococcus pneumoniae (Spn) (Wu 2021). While IL-10 was shown to restrict neutrophil response in Spninfection (Horn 2023), its involvement in immune priming prior to infections remains unclear. We hypothesized that IL-10 regulates lung immune tone in MOC exposure and plays a role in host immune response during Spn infection. Methods We inoculated 12-week-old, C57BL/6 female mice (n = 20) with MOC once (single MOC), 8-week period (chronic MOC, 1 MOC/weekly), or PBS (IACUC s16-00032). We euthanized mice two weeks following the last exposure and processed lung tissues for single-cell RNA sequencing (scRNA-seq). To examine the role of IL-10 in immune priming of MOC, we inoculated 20 mice with single MOC or PBS with IL-10 antibody injection, paired with Spn challenge two weeks after the last inoculation. After 24 hours, we collected lung lavage fluid for bacterial burden and measured total protein for lung injury. Results We found that alveolar and interstitial macrophages were highly abundant in single MOC group compared to chronic MOC and PBS (Figure 1A red and green bars). Following MOC exposure, we identified a subpopulation of macrophages that did not have gene markers Chil3 or Cx3cr1 (Figure 1A blue bar). We identified interstitial and alveolar macrophages using scRNA-seq of which 2.16% of all macrophages were expressing Il-10 (Figure 1B). We investigated the role of IL-10 blockade during MOC (Figure 1C). We found that MOC with IL-10 blockade improved bacterial clearance compared to MOC control mice (Figure 1D). Lower total protein in lung lavage samples of IL-10-blocked MOC samples suggested reduced permeability of alveolocapillary membrane (Figure 1E). These findings align with prior reports that IL-10 production by macrophages is critical to host-pathogen reactions by modulating host response following MOC exposure. Conclusions Our findings provide an integrated framework for understanding the immune priming mechanism of early onset of aspiration by using oral commensals. We explored how IL-10-mediated regulation shapes the immune priming in the lower airways against pathogens. Our ongoing analyses aim to delineate IL-10 interactions with IL-17-expressing cells and signaling networks, such as work by Kim et al. (2025) that determine the outcome of host-microbiota interactions in pneumonia and immune response. This abstract is funded by: Veterans Affairs IK2BX005309-01A2 (BGW); National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, through Grant Award Number KL2TR001446 (BGW); NYU CTSA grant (TL1 TR001447) from the National Center for Advancing Translational Sciences, National Institutes of Health (CJC);CHEST Foundation Research Grant in Chronic Obstructive Pulmonary Disease (BGW); R37 CA244775 (LNS, NCI/NIH); R33 GM147800 (LNS, NIGMS/NIH); U01 AG088351 (LNS, NIA/NIH)