Poster CC15 - Engineering Immune Dysregulation in Acute Respiratory Distress Syndrome

Introduction: Lung inflammation plays a significant role during the onset of respiratory diseases, initiated by various inflammatory stimuli such as bacteria, viruses, mechanical injury, pollution, or chemicals. Acute respiratory distress syndrome (ARDS) is a severe condition characterized by acute lung inflammation and respiratory failure, and results in a hospital mortality rate of approximately 40%.1Immunosuppressive myeloid cells, termed myeloid-derived suppressor cells (MDSCs), play a vital role in shaping immune balance in acute respiratory diseases. MDSCs are involved in the pathogenesis of ARDS due to their contribution to the amplification of pulmonary inflammation and tissue damage. Current therapies utilized to deplete MDSCs, ranging from small molecule inhibitors to monoclonal antibodies (mAbs), encounter challenges in specifically targeting MDSCs, resulting in broad off-target effects and limited efficacy. The development of novel immunomodulatory therapies targeting MDSCs can potentially mitigate disease severity in these conditions. We have developed synthetic nanoparticle antibodies (SNAbs) to target and deplete MDSCs and restore beneficial host immune responses in models of ARDS. SNAbs are multivalent, bifunctional Janus gold nanoparticles (AuNPs) featuring MDSC-targeting ligands on one face and antibody-fragment crystallizable (Fc)-mimicking ligands on the other. By bridging MDSCs with immune effector cells, SNAbs have demonstrated the capability to induce the depletion of MDSCs. We administered SNAbs to ARDS-challenged murine lung cells in vitro and through aerosolized delivery to a mouse model of ARDS. Following flow cytometry analysis and protein analysis, we demonstrated that SNAbs can deplete MDSCs, reduce immunosuppressive mediator (IDO, iNOS, and ARG1) expression, and restore beneficial T cell responses.

Materials and

Methods: SNAbs are fabricated through solid-phase synthesis. Aminomethyl resin was hydrated and reacted with a reducible crosslinker (Sulfo-NHS-SS-biotin) to bind 30-nanometer streptavidin-coated NPs. The crosslinker's disulfide bonds were cleaved with tris(2-carboxyethyl) phosphine (TCEP), releasing Janus nanoparticles with anisotropic surface chemistry, one “face” having free streptavidin for biotin binding and the other with free thiols for maleimide reaction, providing spatially separated conjugation of two different ligands. The domains were functionalized with biotinylated MDSC-targeting ligands, or maleimide functionalized Fc-binding ligands.
We developed a custom nebulizer system for delivering endotoxins for initiating ARDS and for administering therapeutic nanoparticles. This comprehensive system included a nebulizer unit, a control module, custom 3D-printed flexible nose cones, small mouse holder tubes, and a transparent PVC tee. To establish the ARDS model, we administered several combinations of lipopolysaccharides (LPS) and lipoarabinomannan (LAM) via nebulization, testing LPS doses of 2mg/kg and 5mg/kg, and LAM doses of 0.1mg/kg and 1 mg/kg. To evaluate SNAb efficacy in vitro, we collected lungs from ARDS-challenged mice 24 hours post-initial challenge, processing them into a single-cell suspension and treating them with SNAbs at a ratio of 1 million cells to 2.5x1010 particles. SNAbs targeting S100A8/A9, CD124, and CD206 receptors, which are upregulated in MDSCs, were administered, including a combination formulation with equal proportions of each type of SNAb. As controls, we administered PBS or blank gold nanoparticles. Cells were harvested and stained for flow cytometry 24 hours post-SNAb treatment. Following SNAb treatment, cells were harvested and stained for flow cytometry analysis 24 hours later.

Results, Conclusions, and Discussions: ARDS is induced in mice by administrating lipopolysaccharides (LPS), which are cell wall components of gram-negative bacteria. LPS triggers epithelial damage and lung inflammation leading to the recruitment of neutrophils. Additionally, we administer lipoarabinomannan (LAM), a cell wall glycolipid of Mtb. LAM acts as a virulence factor, inhibiting macrophages from killing bacteria and imposing immunosuppression. Together, LPS and LAM can cause damage to the lungs that is both inflammatory and suppressive.
Administering 5 mg/kg LPS and 1 mg/kg LAM via nebulization to C57Bl/6 mice in a total volume of 500uL endotoxin-free water was the most effective method. This combination recruited high frequencies of various cells commonly present during ARDS, including MDSCs, as demonstrated by flow cytometry analysis conducted 24 hours after the initial challenge. This model allows us to study the pathophysiology of ARDS and assess the effectiveness of SNAbs targeting MDSCs and other immune cells implicated in lung injury.
ARDS challenge increased the mean fluorescence intensity (MFI) of the SNAb target receptors (S100A8/A9, CD124, and CD206) on the surfaces of M- and G-MDSCs. Among myeloid cell populations, all 4 of the SNAbs depleted MMDSCs, neutrophils, and interstitial macrophages (IMs) when compared to ARDS groups. Simultaneously, alveolar macrophage (AM) frequencies were higher in SNAb-treated groups than in ARDS alone. We believe neutrophils and IM frequencies were lower in SNAb-treated groups due to the possible reduction of inflammation. These cells are key in the initial immune responses contributing to the cytokine storm and the excessive inflammation associated with ARDS. It is important to note that S100A8/A9, CD206, and F4/80 are markers shared among MDSCs, macrophages, and neutrophils. The intracellular expression of several immunosuppression mediators was observed on CD11b+ cells; however, no significant effects were demonstrated by SNAb-treated groups. This system and experimental approach facilitated the precise initiation of ARDS and the effective assessment of SNAb function, laying the groundwork for targeted therapeutic interventions in acute lung inflammation.

Acknowledgements (Optional): This project is supported by NIH Grant No. R01AI155023, and the Robert A. Milton Endowed Chair funds to KR.