Keywords

Vitamin D; CYP27B1; VDR; CAMP; Crohn's disease; Paratuberculosis

Abstract

Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that Crohn's disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 hours post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78±0.88 and 13.90±3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93±0.28, 1.86±0.27, and 6.34±0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment, and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. In conclusion, this study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.

Completion Date

2024

Semester

Spring

Committee Chair

Naser, Saleh

Degree

Master of Science (M.S.)

College

College of Medicine

Department

Biotechnology

Format

application/pdf

Language

English

Rights

In copyright

Release Date

May 2025

Length of Campus-only Access

1 year

Access Status

Masters Thesis (Campus-only Access)

Accessibility Status

Meets minimum standards for ETDs/HUTs

Restricted to the UCF community until May 2025; it will then be open access.

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