Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits Libby amphibole fiber-induced acute inflammation in mice

Abstract

Background: Exposure to the Libby amphibole (LA) asbestos-like fibers found in Libby, Montana, is associated with inflammatory responses in mice and humans, and an increased risk of developing mesothelioma, asbestosis, pleural disease, and systemic autoimmune disease. Flaxseed-derived secoisolariciresinol diglucoside (SDG) has anti-inflammatory, anti-fibrotic, and antioxidant properties. We have previously identified potent protective properties of SDG against crocidolite asbestos exposure modeled in mice. The current studies aimed to extend those findings by evaluating the immunomodulatory effects of synthetic SDG (LGM2605) on LA-exposed mice.

Methods: Male and female C57BL/6 mice were given LGM2605 via gavage initiated 3 days prior to and continued for 3 days after a single intraperitoneal dose of LA fibers (200 μg) and evaluated on day 3 for inflammatory cell influx in the peritoneal cavity using flow cytometry.

Results: LA exposure induced a significant increase (p < 0.0001) in spleen weight and peritoneal influx of white blood cells, all of which were reduced with LGM2605 with similar trends among males and females. Levels of peritoneal PMN cells were significantly (p < 0.0001) elevated post LA exposure, and were significantly (p < 0.0001) blunted by LGM2605. Importantly, LGM2605 significantly ameliorated the LA-induced mobilization of peritoneal B1a B cells.

Conclusions: LGM2605 reduced LA-induced acute inflammation and WBC trafficking supporting its possible use in mitigating downstream LA fiber-associated diseases.

Summary: Following acute exposure to Libby amphibole (LA) asbestos-like fibers, synthetic SDG (LGM2605), a small synthetic molecule, significantly reduced the LA-induced increase in spleen weight and peritoneal inflammation in C57BL/6 male and female mice. Our findings highlight that LGM2605 has immunomodulatory properties and may, thus, likely be a chemopreventive agent for LA-induced diseases.

Keywords: Asbestos; Autoimmunity; Inflammation; LGM2605; Libby amphibole; Secoisolariciresinol diglucoside.

Figure 1:. Experimental Plan of LA Exposure and LGM2605 Treatment

100 mg/kg LGM2605 (synthetic SDG) or vehicle (saline) was administered via gavage to male and female C57BL/6 (14 weeks old) mice starting 3 days prior to exposure to 200 μg LA via intraperitoneal injection and continued 3 days post-LA treatment (CTL, n=17; LGM2605, n=18; LA, n=17; and LA + LGM2605, n=18). Mice were evaluated at 3 days post-LA exposure for evidence of acute peritoneal inflammation (spleen weights, white blood cell levels, and differential white blood cell influx).

Figure 2:. LGM2605 Ameliorates the LA-Induced Increase in Spleen Weights at 3 Days Post Exposure

Mice were treated with 100 mg/kg LGM2605 or vehicle and exposed to 200 μg LA. Spleens were harvested and weighed at 3 days post-LA exposure. Spleen weight was adjusted for mouse body weight (A). Data are also presented stratified by sex (B). All data are presented as the mean ± the standard error of the mean. # indicates a statistically significant difference (#### p< 0.0001) from CTL and asterisks indicate a statistically significant difference from LA (*** p<0.001 and **** p<0.0001).

Figure 3:. LGM2605 Reduces Peritoneal WBC Levels at 3 Days Post LA-Exposure

Mice were treated with 100 mg/kg LGM2605 or vehicle and exposed to 200 μg LA. PLF was harvested at 3 days post-LA exposure and evaluated for total WBCs (A). Data are also presented stratified by sex (B). All data are presented as the mean ± the standard error of the mean. # indicates a statistically significant difference (#### p< 0.0001) from CTL and asterisks indicate a statistically significant difference from LA (** p<0.01 and **** p<0.0001).

Figure 4:. LGM2605 Inhibits Peritoneal PMN Cell Influx at 3 Days Post-LA Exposure

Mice were treated with 100 mg/kg LGM2605 or vehicle and exposed to 200 μg LA. PLF was harvested at 3 days post-LA exposure and evaluated for PMN cell influx by determining the percentage of Ly6G-positive granulocytes (A). Data are also presented stratified by sex (B). All data are presented as the mean ± the standard error of the mean. # indicates a statistically significant difference (#### p<0.0001) from CTL and asterisks indicate a statistically significant difference from LA (**** p<0.0001).

Figure 5:. LGM2605 Alters Peritoneal Lymphocytes Following LA Exposure

Mice were treated with 100 mg/kg LGM2605 or vehicle and exposed to 200 μg LA. PLF was harvested at 3 days post-LA exposure and evaluated for total lymphocytes (A). Data are also presented stratified by sex for total lymphocytes (B). All data are presented as the mean ± the standard error of the mean. # indicates a statistically significant difference (#### p< 0.0001) from CTL and asterisks indicate a statistically significant difference from LA (* p<0.05).

Figure 6:. LGM2605 Alters Peritoneal Macrophages Following LA Exposure

Mice were treated with 100 mg/kg LGM2605 or vehicle and exposed to 200 μg LA. PLF was harvested at 3 days post-LA exposure and evaluated for total macrophages (A). Data are also presented stratified by sex for total macrophages (B). All data are presented as the mean ± the standard error of the mean. # indicates a statistically significant difference (## p< 0.01 and ### p< 0.001) from CTL and asterisks indicate a statistically significant difference from LA (* p<0.05, ** p<0.01, and *** p<0.001).

Figure 7:. LGM2605 Treatment Does Not Alter Peritoneal B Cells, T Cells, and T Helper Cells Following LA Exposure

Mice were treated with 100 mg/kg LGM2605 or vehicle and exposed to 200 μg LA. PLF was harvested at 3 days post-LA exposure and evaluated for total B cells (A), T cells (B), and T helper cells (C). All data are presented as the mean ± the standard error of the mean. # indicates a statistically significant difference (# p< 0.05) from CTL.

Figure 8:. LA Exposure Decreases Levels of Peritoneal B1a B Cells and B Suppressor Cells

Mice were treated with 100 mg/kg LGM2605 or vehicle and exposed to 200 μg LA. PLF was harvested at 3 days post-LA exposure and evaluated for total B1a B cells (A), alpha-4 integrin levels (C), and total B suppressor cells (E). Data are also presented stratified by sex for total B1a B cells (B), alpha-4 integrin levels (D), and total B suppressor cells (F). All data are presented as the mean ± the standard error of the mean. # indicates a statistically significant difference (#### p< 0.0001) from CTL and asterisks indicate a statistically significant difference from LA (* p<0.05, ** p<0.01, and *** p<0.001).

Figure 8:. LA Exposure Decreases Levels of Peritoneal B1a B Cells and B Suppressor Cells

Mice were treated with 100 mg/kg LGM2605 or vehicle and exposed to 200 μg LA. PLF was harvested at 3 days post-LA exposure and evaluated for total B1a B cells (A), alpha-4 integrin levels (C), and total B suppressor cells (E). Data are also presented stratified by sex for total B1a B cells (B), alpha-4 integrin levels (D), and total B suppressor cells (F). All data are presented as the mean ± the standard error of the mean. # indicates a statistically significant difference (#### p< 0.0001) from CTL and asterisks indicate a statistically significant difference from LA (* p<0.05, ** p<0.01, and *** p<0.001).