Objectives/Goals: Primary sclerosing cholangitis (PSC) manifests with an inflammatory milieu that leads to fibrotic scarring of the liver. Human PSC liver bile ducts are enriched with neutrophils; however, their infiltration and functional role is unexplored. Our goal is to investigate the mechanism and impact of peribiliary neutrophil infiltration observed in PSC. Methods/Study Population: Primary cholangiocytes (bile duct cells) isolated from WT and mouse models of PSC (3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice and Mdr2-/- mice) were analyzed by RNA-sequencing. Immunofluorescence (IF) was performed on liver tissues from PSC patients and mouse models of PSC for markers of bile ducts and neutrophils (KRT19 and MPO). Intrahepatic leukocytes (IHL) isolated from mice livers were evaluated for neutrophil abundance and activation state. Anti-Ly6G antibody-mediated neutrophil depletion in Mdr2-/- mice was analyzed by IF, histology, and cytometry by time-of-flight (CyTOF). Cholangiocytes stimulated with TNFα (to induce an inflammatory phenotype) were analyzed for neutrophil chemoattractants with genetic and pharmacological interventions. Results/Anticipated Results: RNA-seq analysis of primary cholangiocytes from PSC mouse models revealed enrichment in inflammatory and neutrophil degranulation pathways. Flow cytometry and RT-PCR analysis revealed an increase in the neutrophil population in PSC mice with activated phenotype. Peripheral depletion of neutrophils in Mdr2-/- mice alleviated liver injury and inflammation, along with a reduction in peribiliary neutrophil infiltration and attenuated bridging fibrosis. CyTOF analysis showed a significant reduction in CD8+ T cells upon neutrophil depletion, implying neutrophils sustain CD8+ T cells in PSC liver. Mechanistically in cholangiocytes, TNFα mediates expression of neutrophil chemoattractants, CXCL1 and CXCL8, through the cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING) pathway. Discussion/Significance of Impact: Our findings suggest that activation of the STING pathway in cholangiocytes in cholestatic liver disease triggers an immune response resulting in peri-portal neutrophil infiltration via CXC chemokines. The sustained presence of these activated neutrophils engages the adaptive immune system to perpetuate the inflammation and fibrosis seen in PSC.

Objectives/Goals: Depression is common among people living with HIV (PLWH). This study explored the link between reduced metacognitive awareness and depression in PLWH. It utilized a positive emotion regulation task to compare brain activation during viewing versus upregulating positive emotions. Methods/Study Population: Depressed PLWH (N = 24; mean age  =  53; HAM-D mean  =  19) participated in an emotion regulation task while blood oxygen-level-dependent (BOLD) responses were recorded. In the emotional regulation task, participants were shown the International Affective Picture System (IAPS) a series of positive, negative, and neutral images. Participants were asked to view these images and given instructions to either negatively reappraise (RN) or positively reappraise (RP). In the RP condition, participants were no longer shown the image and asked to upregulate their positive emotional responses associated with it. Ten onset times were included for each trial. Results/Anticipated Results: A one-sample t-test was conducted to analyze contrasts between reappraisal of positive images and viewing positive images (RP > VP). Results showed significantly greater activation in the posterior cingulate and angular gyrus during the RP condition (peak MNI: 18, -52, 34; p < 0.001, uncorrected, k > 10 voxels). In comparing the reappraisal of negative images to viewing negative images (RN > VN), there was increased activation in the right supramarginal gyrus (peak MNI: 50, -28, 22; p < 0.001, uncorrected, k > 10 voxels). When contrasting the reappraisal of positive to negative images (RP > RN), BOLD signals were higher in the left dorsolateral prefrontal cortex (peak MNI: 40, -38, 32; p < 0.001, uncorrected, k > 10 voxels). Discussion/Significance of Impact: Findings underscore that depressed PLWH demonstrates BOLD responses in brain regions linked to appetitive motivation and meta-cognitive awareness during the RP condition which demands more executive resources among those with depression, highlighting the complexity of emotional regulation in this population.