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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster H18 - Investigating Changes in Lymphatic Endothelial Cell Junction Expression and LEC-mediated Immunity Post Exposure to Particulate Matter

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Small particulate matter (PM) consists of fine, carbon-based particles from sources like wildfire smoke and exhaust fumes. Urban residents are regularly exposed to PM via inhalation, which has been linked to asthma, reduced lung function, heart problems, and premature death. PM can accumulate in the lymph nodes, indicating it diffuses through the lungs and into the lymphatic system.
Lymphatic vessels are made up of lymphatic endothelial cells (LECs) which modulate immunity by harboring and trafficking immune cells. LECs have continuous zipper-like and discontinuous button-like junctions that regulate fluid flow and cell passage. Abnormal LEC junction morphology can cause fluid leakage, dysfunctional immune responses, and is associated with lung adenocarcinoma and COPD. Studies show that mice with lymphatic dysfunction have heightened inflammatory responses to stimuli.
LECs also have immunomodulatory capabilities, and are involved in immune cell trafficking, signaling, and activation. PM exposure in keratinocytes increases the expression of inflammatory cytokines like IL-1β and immune cell-modulating cytokines like IL-16. LECs play a crucial role in antigen presentation via MHC-II and may suppress the immune system in tumors, facilitating cancer proliferation. However, the effects of PM on lymphatic junction permeability and immune response modulation in LECs are not well studied. This study examines the effects of PM on LEC-mediated immunomodulation and LEC junction formation.

Materials and

Methods:  Human dermal LECs were exposed to physiological concentrations of National Institute of Standards and Technology (NIST) standardized urban particulate matter (0, 10, 20, and 50 µg/mL) for 48 hours. A transport assay was performed by seeding LECs on the bottom of a semipermeable transwell, exposing them to 20 µg/mL PM, and measuring the rate of transport across the LEC monolayer (Figure 2A). The LEC monolayer was then stained for DAPI and VE-cadherin. LECs, known to differentially express cytokines in the presence of IFN-γ, were divided into four treatment groups: basal without PM treatment, basal with PM treatment, IFN-γ-stimulated without PM treatment, and IFN-γ-stimulated with PM treatment. PM-treated LEC monolayers were immunostained for junction proteins VE-cadherin (adherens) and ZO-1 (tight). Confocal images were taken and quantitatively assessed by the Junction Analyzer Program (JAnaP), a Python-based program that characterizes cell-cell junctions as either normal continuous junctions or abnormal punctate and perpendicular junctions.

RT-qPCR was used to assess the expression of podoplanin, a transmembrane receptor glycoprotein highly expressed in LECs and involved in cell migration. Immunomodulatory molecules and cytokines were selected using a single-cell RNA sequencing dataset and validated through primary literature to confirm that LECs secrete the chosen cytokines. RT-qPCR will also be performed to assess the expression of immune cell activation/repression molecules (PD-L1, CSF-1, IL-6, IL-8, and MHC-II) and immune cell homing/migration molecules (CXCL10, β-catenin, CCL21, and CXCL13). β-catenin is involved in forming cell-cell adhesion complexes and is necessary for cells to migrate through lymphatic vessels.

Results, Conclusions, and Discussions: Immunofluorescent images of LECs exposed to PM showed formation of abnormal spindle-like junctions (Figure 1B). This suggests that PM exposure in LECs may lead to a leakier junction phenotype and decreased monolayer integrity. Transport data support this hypothesis as exposure to PM at concentrations of 10 and 20 µg/mL showed a significant increase in transport efficiency across the LEC monolayer when compared to the control (Figure 1C).
Brightfield images of PM-treated LECs show that LECs take up progressively more PM with increasing treatment concentration(Figure 2 A-D). The increased uptake upon PM exposure suggests that there may be an increase in permeability of LEC monolayers, which aligns with previous literature showing that PM can accumulate in lymph nodes via lymphatic vessels. This also suggests that PM may also stimulate an inflammatory response in LECs, as VE-cadherin expression can become disjointed in response to inflammatory stimuli.
Based on the increase in transport when LEC monolayers are exposed to PM, we expect to see a decrease in continuous junction expression upon treatment with PM, which correlates to a leakier lymphatic vessel phenotype. Previous studies have shown that exposure to PM induces an inflammatory immune state. Therefore, we expect to find that LECs exposed to PM will produce more inflammatory cytokines like IL-6 and increase expression of immune cell homing and activating markers like MHC-II. Understanding how LECs influence this immune response to PM will be critical to developing strategies for mitigating its harmful effects in tissues
Exposure to PM leads to lymphatic uptake, causes the formation of abnormal junctions in LEC monolayers, and increases transport across these monolayers, suggesting weakened barrier integrity. PM may also induce an inflammatory response in LECs. These findings have implications for understanding how air pollution and PM affects LEC’s barrier function, junction morphology, and immunomodulatory capabilities which will be important for developing potential treatments for conditions exacerbated by air pollution, like asthma.

Acknowledgements (Optional):