(A-10) The novel marine echinoderm-derived exosomes regulate autoimmune disease

Introduction:: Atopic dermatitis (AD) is a chronic skin condition that results in dry, itchy, and inflamed skin. Genetic and environmental factors cause it and can affect children and adults. The current treatment of atopic dermatitis aims to relieve symptoms. Typical treatment includes topical corticosteroids or immunomodulators to reduce inflammation and to take oral medications such as antihistamines or antibiotics. To address the insufficient, stem cell-derived exosomes were used. Exosome therapy for AD is an emerging experimental treatment, but research is still in the early stages. This study aims to explain atopic treatment using marine echinoderms-derived exosomes. The marine echinoderms composed of star fish, sea urchin, and sea cucumber. The marine echinoderms have unique biological behavior such as regeneration ability and immune system. In particular, the sea cucumber’s immune system is more complex and robust than mammals. Therefore, we predict that a sea cucumber-derived exosome (SEVs) can alleviate an anti-inflammatory environment. Set the hypothesis that marine-echinoderm exosomes had complex immune systems that can regulate the atopic autoimmune pathway.


Materials and Methods::

• Marine-echinoderm exosome isolation

To isolate the SEVs, the sea cucumber extracellular-matrix (SECM) was treated with a disaggregating solution containing PBS and collagenase. The digested SECM was subjected to a continuous centrifugation process to remove the ECM debris. The supernatant was filtered and then treated precipitation solution. (Process was applied for a patent, so couldn't disclose the details)

• Marine-echinoderm characterization

The SEVs were visualized using FE-SEM and TEM. The SEV was fixed with paraformaldehyde and washed. The SEV suspension was dropped on a cover glass or carbon-coated grid. The SEVs were diluted in PBS and determined using DLS. To determine the protein size distribution, and SDS-PAGE were performed. Purified RNA was sent to Macrogen (Seoul, Korea) to analyze the profile of small RNAs using a Bioanalyzer.

• Anti-inflammation analysis

To test the anti-inflammatory effects, macrophages were treated with SEV suspension (10 μg/mL) in FBS-free RPMI-1640 medium with LPS (50 ng/mL). After 48h, real-time PCR, and immunocytochemistry (ICC) experiments were conducted.

• Gene screening and analysis

The SEV was sent to Macrogen to analyzie mRNA expression.

• Atopic dermatitis in vivo analysis

AD were induced by using DNCB. Briefly, the upper mice backs were shaved with a clipper. After 24 h, 150 μl of 1% DNCB in acetone: olive oil mixture (4:1) was topically applied on days 1 through 5. Later, the same dose of 0.2% DNCB and SEVs was applied thrice a week for four weeks. After sacrifice, blood, skin, and spleen were extracted. Subsequently, collected tissues were analyzed by ELISA, real-timePCR, and histology.

Results, Conclusions, and Discussions::

• Marine echinoderm-derived exosome characterization

SEM and TEM data show round shape particles and the size looks around 200nm. In addition, the SEV size distribution result shows that almost particles have 150-200nm range. Moreover, SEV’s cargos were analyzed. The SDS-PAGE result shows many kinds of protein they have. Furthermore, the SEV gene cargo length was determined, and the SEV’s RNA cargos have an 18-150nt range. In particular, the peaks of 22 and 67nt were high intensity. The genetic material range of 18-22nt in length is known as microRNA, and the range of 60-150nt is known as transfer RNA. We expected that the miRNAs inside SEV would affect to regulate the immune system because previous research was reported about microRNA regulating gene expression.

• Anti-inflammation analysis

To check the anti-inflammatory effect, LPS and LPS+SEV were treated with macrophage. Real-timePCR results, the SEV treatment group showed pro-inflammatory cytokine suppressions and anti-inflammatory cytokines expression increased. Moreover, ICC results showed that SEV could suppress the macrophage phenotype-1 marker.

• Gene screening and analysis

Micro-array results showed normal, inflammation, and inflammation+SEV treatment groups have different gene expression trends. Compared with inflammation and SEV treatment, the five genes with the most significant difference expressions are down-regulation (IFI-16, CCL4, SLFN4) and up-regulation (Mt2, Abcc5). Subsequently, GO and KEGG were conducted. The analysis showed that SEV involved immune regulation such as NOD-like receptor, IL-17, and Toll-like receptor signaling pathway. Therefore, the SEV has IFI-16 gene suppress ability. We applied it to treat atopic dermatitis. Previous research reported that the IFI-16 gene is a cue signal of the inflammasome and contributes to psoriasis.

• Atopic dermatitis in vivo analysis

AD induced mice were treated using SEV (0, 1, and 10μg) for three weeks. During analysis, skin thickness and AD scoring decreased in the SEVs-treated group. ELISA, real-timePCR, and histological data showed that dose-dependently AD gene and protein factors were suppressed. Furthermore, the toluidine-blue test result showed that the number of mast cells was reduced.

• Conclusions

We found novel marine-derived exosomes. They have anti-inflammatory effects and could alleviate AD factors. Therefore, the SEVs could be promising therapeutic candidates for AD


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