Linden, David

Background: Diabetic gastroparesis (DG) is associated with a loss in anti-inflammatory muscularis macrophages (MMs), contributing to delayed gastric emptying (DGE). Lyve1 macrophages, known for their anti-inflammatory phenotype, are involved in lipid metabolism across organs. While diabetes is typically associated with increased lipid levels, the dependence of lipid handling on altered function of Lyve1+ MMs in the stomach remains unknown. Aim: To investigate lipid accumulation in MMs and test the effect of palmitate and CD36 on Lyve1+ macrophages. Methods: FACS sorted CD45+ cells were isolated from the gastric muscularis of non-diabetic mice and analyzed using 10X Genomics Cellranger and Seurat package. Gene Set Enrichment Analysis was performed to identify enriched pathways. Separately, the cells underwent flow cytometry to identify Lyve1+/- MMs then determining lipid accumulation by LipidTox intensity. RAW 264.7 macrophage cells were cultured with macrophage colony stimulating factor (M-CSF), IL-4, and dexamethasone for 7 days to induce Lyve1 expression. RNA was collected from cells treated with palmitate and a CD36 inhibitor, Sulfosuccinimidyl oleate (SSO), for RT-qPCR. Lyve1+ and Lyve1- macrophages treated with 300µM palmitate overnight were collected for lipidomic analysis. Untargeted lipidomic analysis was done with Thermo Orbitrap Exploris 120, and data were analyzed using principal component analysis for group clustering and unpaired t-tests for differential analysis. Results: Unbiased clustering analysis revealed 6 transcriptionally distinct gastric MM populations, one of which was notably enriched in Lyve1 expression. Pathway analysis identified that Lyve1 enriched cluster was associated with lysosomal pathways and receptors involved in lipid uptake, such as CD36, as well as anti-inflammatory markers such as HO1 and CD206. Flow cytometry quantification showed that Lyve1+ MMs had higher accumulation of lipids compared to Lyve1- MMs (10.1±0.7 vs 2.9±0.2 mean fluorescence intensity/cell count, p=0.0002). RT-qPCR validated that Lyve1+ RAW 264.7 macrophages exhibited significantly higher expression of Lyve1 (6.95 times fold change, p=0.023) and CD36 (3.27 times fold change, p=0.011) compared to Lyve1- macrophages. Post palmitate treatment, Lyve1+ macrophages had increased expression of the pro-apoptotic gene caspase-3 (12.15-times fold change, p=0.033), as well as the fatty acid oxidation marker ACADM (4.98 times fold change, p=0.045). Notably, these differences were not found in the presence of SSO. Lipidomic analysis identified 171 differentially expressed lipids, particularly increased levels of ceramides and diacylglycerols in Lyve1+ macrophages compared to Lyve1- macrophages when both are treated with palmitate. Conclusions: Lyve1+ macrophages preferentially uptake lipids compared to Lyve1- macrophages consistent with increased expression of CD36. Lipid treatment of Lyve1+ macrophages increases expression of apoptosis and oxidation markers in a CD36-dependent manner that may lead to cell loss. These mechanisms may contribute to Lyve1+ MMs loss in DG. Funding: DK127992; DK129297; DK115255; ANMS Young Investigator grant; AGA grant#36; P30DK084567; Robert and Arlene Kogod Center on Aging Fundamental Mechanisms of Aging Award. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract Preterm infants can develop airway hyperreactivity and impaired bronchodilation following supplemental O 2 (hyperoxia) in early life, making it important to understand mechanisms of hyperoxia effects. Endogenous hydrogen sulfide (H 2 S) has anti‐inflammatory and vasodilatory effects with oxidative stress. There is little understanding of H 2 S signaling in developing airways. We hypothesized that the endogenous H 2 S system is detrimentally influenced by O 2 and conversely H 2 S signaling pathways can be leveraged to attenuate deleterious effects of O 2 . Using human fetal airway smooth muscle (fASM) cells, we investigated baseline expression of endogenous H 2 S machinery, and effects of exogenous H 2 S donors NaHS and GYY4137 in the context of moderate hyperoxia, with intracellular calcium regulation as a readout of contractility. Biochemical pathways for endogenous H 2 S generation and catabolism are present in fASM, and are differentially sensitive to O 2 toward overall reduction in H 2 S levels. H 2 S donors have downstream effects of reducing [Ca 2+ ] i responses to bronchoconstrictor agonist via blunted plasma membrane Ca 2+ influx: effects blocked by O 2 . However, such detrimental O 2 effects are targetable by exogenous H 2 S donors such as NaHS and GYY4137. These data provide novel information regarding the potential for H 2 S to act as a bronchodilator in developing airways in the context of oxygen exposure.