Poster CC1 - In Vitro Modeling of Systemic Lupus Erythematosus Associated Small Vessel Vasculitis for Characterization of Monocyte-Endothelial Cell Interactions

Introduction: Systemic Lupus Erythematosus (SLE) affects over a million people in the US [1]. SLE is a chronic, systemic autoimmune disease that dysregulates both the adaptive and innate immune systems. Approximately 9 in 10 SLE diagnoses are women of reproductive age [1]. SLE associated inflammation of vasculature, known as Lupus Vasculitis (LV), occurs in approximately 50% of SLE cases [2]. LV can afflict vessels of any size, although small vessel involvement is the most common [3]. Atypical microvasculature formation associated with LV results from monocyte modulation and the subsequent recruitment of immune cells into a microvascular environment [2,4].
Monocytes play a crucial role in vasculature inflammation through interactions with endothelial cells, crucial cells lining blood vessels. Endothelial cells produce chemokines and adhesion molecules in response to inflammatory stimuli. This promotes the recruitment and adhesion of monocytes to the endothelium. Presently, the interactions between monocyte and endothelial cells in SLE are not well characterized. There are no current in vitro models of small vessel vasculitis in SLE and clinical presentation is not quantified.
In response to this limitation, we have created a microphysiological system that mimics capillary flow to introduce a preclinical model of small vessel vasculitis in SLE.
We hypothesize that SLE-like monocytes will alter the phenotypic behavior of endothelial cells causing inflammation in a microphysiological system mimicking capillary flow.

Materials and

Methods: A Polydimethylsiloxane microfluidic device was prepared with 200μm acupuncture needles inserted through the gel reservoir. The gel reservoir was then injected with 20μL of collagen gel and incubated at 37°C for 30 minutes. Following incubation, the needles were removed, and the openings were sealed. The top two media inlets were then filled with 45μL each of endothelial cell suspension at 1M/mL. The device was inverted then incubated for 5 minutes allowing adhesion of endothelial cells into the channel within the collagen gel as the cell suspension flows through. The cell suspension was then aspirated and replaced with a new 45μL of suspension. This device was then incubated for 5 minutes before cell suspension was removed and replaced with media in media inlets (see Figure 1:A for workflow). The device was incubated on a rocker for 72hrs with daily media changes. Following the 72hrs, the media was aspirated and 45μL of cell suspension of 1.5M/mL SLE- stimulated macrophages are placed into the top two media inlets. These devices are incubated on a rocker to flow through the endothelial cell lined channel for thirty minutes. Following incubation, the cell suspension was removed and replaced with medium in all four channels and incubated for 24hrs. Following incubation, six devices went through collagenase cell-extraction for RT-PCR evaluating inflammatory markers associated with LV. A dextran diffusion assay was then conducted on six devices to investigate endothelial barrier integrity. The remaining six devices are used for immunocytochemistry to investigate the same markers evaluated in RT-PCR.

Results, Conclusions, and Discussions: This work creates the first model of small vessel vasculitis in SLE. The endothelial vessel within this system allows for monocyte flow and adhesion (Figure 1: E,D). In the optimization of this system, two monocyte densities were evaluated for flow through the vessel. The 1.25M/mL density was chosen as it is closer to physiologically relevant to flow rate levels of monocytes in whole blood and both densities qualitatively demonstrated adequate levels of monocytes that enter the channel (Figure 1: B,C).
To establish proof of concept, the RAWs were treated with resiquimod (R848). R848 is a TLR7/8 agonist, is used to induce SLE in animal models6. Because of such, R848 serves as method to stimulate monocytes and macrophages into a SLE-like state.
Our results show that the R848 treated macrophage groups have elevated levels of inflammatory cytokines IL6 and TNF. Additionally, the endothelial cells had increased levels of ICAM1 and E-Selectin, as expected to be seen with endothelial inflammation. Elevated levels of IL6 and TNF aligns with what is seen in serum levels of patients with active SLE. These markers were also investigated and validated through immunocytochemistry. Additionally, the dextran diffusion assay demonstrated increased vessel permeability amongst the R848 treated groups, indicating an inflamed endothelium with decreased function. These results validate the use of this system for investigation into myeloid-endothelial cell interactions in LV.
Future applications with SLE monocytes will provide opportunities for interrogation into the atypical behavior and interactions between these cells. Furthermore, the device has the potential of unveiling components of SLE heterogeneity through the use of monocytes derived from patient of specific demographic backgrounds. Additionally, there is potential for use of primary dermal microvascular endothelial cells to be used, introducing even greater levels of translatability from the in vitro results given the prevalence of dermal inflammation in SLE. Future work will also apply this model to investigate therapeutic targets to monocyte-endothelial behaviors in the context of LV.

References: [1]Lupus facts and statistics. Lupus Foundation of America. (2023) [2]Leone P, et al. Biomedicines. (2021) [3]Barile-Fabris L, et al. Curr Rheumatol Rep. (2014) [4]Chunyan Ma, et al. Clinical Immunology, (2019).

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