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Cathelicidin/LL-37 levels in 24 hours Ethanol, Dially Disulfide Exposed Pulmonary Alveolar Epithelial Cells

1,2T. Dew, 1,3H. Hottor, 1,3O. Ogunsakin, 1,3M. McCaskill 1Emerging Scholars Environmental Health Sciences Academy 2J.S Clark High School 3Department of Global

Environmental Health Sciences, Tulane University SPHTM

RESULTS

DISCUSSION AND CONCLUSION

REFERENCES

INTRODUCTION

METHODS ABSTRACT

Vitamin D is a fat-soluble vitamin that is supplied in the diet in foods like fatty fish, liver oils, and dairy products such as milk. Humans receive an adequate amount of vitamin D when exposed to the sun, as this vitamin is produced endogenously when ultraviolet B energy comes in contact with the skin which initiates the synthesis of vitamin D3 from 7 dehydrocholesterol. The formed vitamin D3 then binds to the vitamin D binding protein and is transported to the liver. In the hepatic system, the vitamin D3 undergoes metabolic conversion to 25 - hydroxyvitamin D[25(OH)D3] by the enzyme 25-hydroxylase. The formed vitamin D[25(OH)D3 is then transported into the renal system and is converted by 1,25 - hydroxylase to 1,25- hydroxyvitamin D[(OH)2D3], the active form of vitamin D. This conversion into active vitamin D is mediated by phase 1 metabolizing enzyme CYP27B1. Consequently, depletion in the levels of vitamin D or disruption of its pathway by agents such as alcohol has resulted in various deleterious health outcomes, including low levels of pulmonary anti-microbial peptide(LL-37), pulmonary bacteremia, and pneumonia. Organ sulfur compounds like DADS, found in garlic, have been shown to inhibit CYP2E1-mediated bioconversion in the pulmonary system via its chemo preventive properties.

This work was supported by the Gulf Region Health Outreach Program (GRHOP) which is funded from the Deepwater Horizon Medical Benefits Class Action Settlement approved by the U.S. District Court in New Orleans on January 11, 2013. Thanks to JD and Mama.

ACKNOWLEDGEMENT

• Janssens, W., Lehouck, A., Carremans, C., Bouillon, R., Mathieu, C., & Decramer, M. (2009). Vitamin D beyond bones in chronic obstructive pulmonary disease: Time to act. American Journal of Respiratory and Critical Care Medicine, 179(8), 630-636

• Kent, J. C., Devlin, R. D., Gutteridge, D. H., & Retallack, R. W. (1979). Effect of alcohol on renal vitamin D metabolism in chickens. Biochemical and Biophysical Research Communications, 89(1), 155-161

• Larrick, J. W., Hirata, M., Zhong, J., & Wright, S. C. (1995). Anti-microbial activity of human CAP18 peptides. Immunotechnology, 1(1), 65-72.

• Lebowitz, M. D. (1981). Respiratory symptoms and disease related to alcohol consumption. The American Review of Respiratory Disease, 123(1), 16-19

• Leevy, C. M., & Moroianu, Ş A. (2005). Nutritional aspects of alcoholic liver disease. Clinics in Liver Disease, 9(1), 67-81

Figure 5. 25(OH) D levels in 24 hour ethanol treated hPAE cells. * p< 0.05 as compared to control.

Figure 6. 25(OH) D levels in 24 hour ethanol treated hPAE cells. * p< 0.05 as compared to control.

Figure 7. Cathelicidin levels in 24 hour ethanol and DADS co-exposed hPAE cells *p<0.05 as compared to the control, ~ p<0.05 as compared to EtOH 80mM.

Figure 4. 25(OH) D, and 1,25 (OH)2 D levels in BALF of Alcohol Use Dependent Patients.

CELL CULTURE & SAMPLE ANALYSIS: The human pulmonary alveolar epithelial cells (hPAE) were

purchased from Sciencell Research Laboratories and was grown in recommended media, AEPiCM catalog #3201. The cells were seeded in 25 cm2 Tissue Culture flask and when near confluence were treated with ethanol and diallyl disulfide as shown in the dosing schedule. (Table 1). Cells were then lysed and the homogenate was prepared for assay.

• Human Cathelicidin/LL-37 (Hycult Biotech) was utilized to quantify homogenate levels of treated hPAE cells, Human BALF and blood serum.

• 25(OH) Vitamin D EIA immnmuodiagnosticsystems (ids) was utilized to quantify inactive vitamin D in treated hPAE cells, Human BALF, and blood serum.

After dosing the appropriate cells and incubating them for twenty four hours, we found that levels of cathelicidin were lowest in flasks dosed only with ethanol at twenty five and eighty millimolar. Compared to the ethanol control groups that were treated, the control group had a higher amount of cathelicidin. The treatment group that contained eighty millimolars of ethanol and DADS had a significantly higher amount of the antimicrobial peptide. After gathering the data on the amount of antimicrobial peptides, Vitamin D in the serum were quantified. The control groups (cells with only medium and cells with medium and DADS) had the highest amounts of Vitamin D in them. The DADS control had a higher amount than the medium control. The ethanol control groups of twenty-five and fifty millimolars had the lowest amount Vitamin D.

Approximately more than 5% of the United States population consumes alcohol in excessive amounts. Extensive research and published works have shown the numerous deleterious effects of alcohol on the brain, liver, and kidney. Alcohol has also been associated with an increase in the severity of respiratory infections among sensitive populations. However, the mechanism by which this occurs is hazy and requires more research in human population. One of the ways ethanol affects vitamin D’s impact in the pulmonary system is through the disruption of vitamin D metabolism and pathways. Subsequently, this inhibits the activation of the protective anti-microbial peptides (Cathelicidin/LL-37) by vitamin D. This study examines the effects of ethanol on vitamin D pathways and antimicrobial peptides (Cathelicidin/LL -37) over a span of 24 hours in human alveolar cells and Bronchoalveolar lavage (BAL) fluids from patients with alcohol use disorder

Figure 3. 25 (OH) D levels in Alcohol Use Dependent Patients

Figure 2.

According to our results, we can conclude that alcohol reduces the levels of vitamin D in the alveolar epithelium. Consequently, this affects the activation of antimicrobial peptides (Cathelicidin/LL-37) and immune cells mediation in the pulmonary system. A low level of LL-37 in individuals with alcohol use disorder exposes to increased frequency and severe form of respiratory infections. In addition, our results showed that DADS has the potential to recover the inhibitory effect of alcohol on the pulmonary epithelia cells. Because we were unable to show the effects of alcohol on active vitamin D, future research will focus on eliciting the overall impact on vitamin D metabolism and antimicrobial peptide among sensitive population