(M1130-07-37) Inflammatory Response to Lipopolysaccharide-Induced Injury and Treatment with Extracellular Matrix-Derived Nanoparticles

Purpose: Acute Respiratory Distress Syndrome (ARDS) is a severe form of acute lung injury (ALI) and is associated with significant morbidity and mortality. It is characterized by fluid accumulation in the lung, gap formation in endothelial lung tissue, and rupture of the alveolar capillary barrier ¹. Various causes can lead to ARDS, including pneumonia (bacterial or viral), trauma, and extra-pulmonary diseases like sepsis and pancreatitis ². The current treatment options include respiratory support via mechanical ventilation and management of the symptoms of respiratory failure, highlighting a need for more therapeutic modalities ³. Our study employs xenographic, organ-specific extracellular matrix (ECM), fabricated as electrically charged nanoparticles (ECM-NPs). ECM consists of organ-specific proteins and growth factors that have been demonstrated to be anti-inflammatory and pro-regenerative by previous studies and could be advantageous for treating ARDS ^4,5. ECM may be therapeutically delivered through inhalation of solubilized suspension or micro-sized inhaled particles; however, organ-specific solubilized inhaled nanoparticles are a novel treatment ⁶. Lipopolysaccharide (LPS) or endotoxin, found in the outer membrane of gram-negative bacteria, is commonly administered to mice either directly into the airways (pulmonary insult) or systemically (extrapulmonary insult) for the induction of ALI ^2,7. We hypothesize that treatment with inhaled ECM-NPs will improve the inflammatory response caused by LPS-induced acute lung injury in mice.

Methods: Twelve week old C57BL/6 mice (male and female) were anesthetized. Following intratracheal intubation, 0.9% saline or LPS (2 mg/kg) were administered. After a time gap of 5 minutes, 50 ul 0.9% saline or ECM nanoparticles (12.5 mg/ml) were administered, and the mice were allowed to recover. 24 hours after LPS dosing, the mice were mechanically ventilated for 10 minutes on non-injurious settings to quantify lung mechanics. Bronchoalveolar lavage fluid (BALF) was analyzed for protein content and inflammatory cytokine levels.

Results: Sex-based differences were observed in the inflammatory response to LPS and the ECM-NP treatment. Compared to the control, significantly higher total protein content (Fig. 1A) and IL-6 levels (Fig. 2A) were observed in the BALF of males exposed to LPS but received no ECM treatment. Similarly, LPS exposure led to a significant increase in the total protein content of the BALF for female mice that did not receive ECM treatment (Fig. 1B). Among the mice exposed to LPS that did not receive the ECM-NP treatment, male mice had significantly higher levels of total protein (Fig. 1C) and IL-6 (Fig. 2C) in the BALF than female mice. Upon treatment with ECM-NPs, female mice had significantly lower levels of total protein content in BALF as compared to mice treated with saline after LPS exposure (Fig. 1B). LPS exposure and subsequent treatment with ECM-NPs did not significantly affect the IL-6 levels in the BALF of female mice (Fig. 2B).

Conclusion: The males responded more strongly to LPS stimulation, as demonstrated by significant increases in the BALF's total protein and IL-6 content. This correlates with previous studies demonstrating that male mice exhibit a stronger immune response to LPS injury than female mice; however, sex-based differences in response to ECM-NPs have not been explored⁸. A significant reduction in the total protein content in the BALF was observed in female mice after ECM-NP treatment. Further studies to elucidate the mechanism behind cell response to ECM treatment after LPS stimulation are planned with the Calu 3 cell line, a human adenocarcinoma cell line used to model the airway. Further steps for the current study include dose-adjustment of the ECM-NPs, to determine if body weight-related variance in the dose was responsible for sex-based disparities in response. Additional studies assessing different dosing intervals could also help guide future work.

References: (1) Matthay, M. A.; Ware, L. B.; Zimmerman, G. A. The Acute Respiratory Distress Syndrome. J Clin Invest 2012, 122 (8), 2731–2740.
(2) Domscheit, H.; Hegeman, M. A.; Carvalho, N.; Spieth, P. M. Molecular Dynamics of Lipopolysaccharide-Induced Lung Injury in Rodents. Front. Physiol. 2020, 11.
(3) Pierrakos, C.; Karanikolas, M.; Scolletta, S.; Karamouzos, V.; Velissaris, D. Acute Respiratory Distress Syndrome: Pathophysiology and Therapeutic Options. J Clin Med Res 2012, 4 (1), 7–16.
(4) Link, P. A.; Ritchie, A. M.; Cotman, G. M.; Valentine, M. S.; Dereski, B. S.; Heise, R. L. Electrosprayed Extracellular Matrix Nanoparticles Induce a Pro‐regenerative Cell Response. J Tissue Eng Regen Med 2018, 12 (12), 2331–2336.
(5) Hocking, D. C. Therapeutic Applications of Extracellular Matrix. Advances in Wound Care 2015, 4 (8), 441–443.
(6) Manni, M. L.; Czajka, C. A.; Oury, T. D.; Gilbert, T. W. Extracellular Matrix Powder Protects Against Bleomycin-Induced Pulmonary Fibrosis. Tissue Engineering Part A 2011, 17 (21–22), 2795–2804.
(7) Tsikis, S. T.; Fligor, S. C.; Hirsch, T. I.; Pan, A.; Yu, L. J.; Kishikawa, H.; Joiner, M. M.; Mitchell, P. D.; Puder, M. Lipopolysaccharide-Induced Murine Lung Injury Results in Long-Term Pulmonary Changes and Downregulation of Angiogenic Pathways. Sci Rep 2022, 12 (1), 10245.
(8) Cai, K. C.; van Mil, S.; Murray, E.; Mallet, J.-F.; Matar, C.; Ismail, N. Age and Sex Differences in Immune Response Following LPS Treatment in Mice. Brain, Behavior, and Immunity 2016, 58, 327–337.

Acknowledgements: This abstract is funded by NIH R21 HL146250-02

Figure 1: ECM-NP treatment reduces total protein content in the BALF of female mice after LPS exposure, with sex-based differences in response to both LPS and ECM-NP treatment. Total protein present in lavage fluid of A) male and B) female mice and, C) sex- based differences (n = 4, 6, 6). Data shown are mean ± SD. One-way ANOVA and Tukey comparisons employed for statistical analysis. * p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2: Sex-based differences in IL-6 levels present in the BALF, in response to both LPS and ECM-NP treatment. IL-6 present in lavage fluid of A) male and B) female mice and, C) sex based differences (n = 4, 6, 6). Data shown are mean ± SD. One-way ANOVA and Tukey comparisons employed for statistical analysis. * p < 0.05, **p < 0.01, ***p < 0.001.