Folate and Vitamin B(12) Deficiency Exacerbate Inflammation during Mycobacterium avium paratuberculosis (MAP) Infection

Folate and vitamin B(12) deficiency is highly prevalent among Crohn’s disease (CD) patients. Furthermore, CD pathology can be mediated by Mycobacterium avium subsp. paratuberculosis (MAP) infection. However, the direct effect of folate (B(9)) and cobalamin (B(12)) deficiency during MAP infection remains uncharacterized. This study investigates how folate and B(12) deficiency impedes macrophage apoptosis and exacerbates the inflammation in macrophages infected with MAP isolated from CD patients. Accordingly, we measured folate and B(12) in ex vivo plasma samples collected from CD patients with or without MAP infection (N = 35 per group). We also measured the expression of the pro-inflammatory cytokines IL-1β and TNF-α, cellular apoptosis and viability markers, and bacterial viability in MAP-infected macrophages cultured in folate and B(12) deficient media. We determined that MAP-positive CD patients have significantly lower plasma folate and B(12) in comparison to MAP-negative CD patients [414.48 ± 94.60 pg/mL vs. 512.86 ± 129.12 pg/mL, respectively]. We further show that pro-inflammatory cytokines IL-1β and TNF-α are significantly upregulated during folate and vitamin B(12) deprivation following MAP infection by several folds, while supplementation significantly reduces their expression by several folds. Additionally, depletion of folate, B(12), and folate/B(12) following MAP infection, led to decreased macrophage apoptosis from 1.83 ± 0.40-fold to 1.04 ± 0.08, 0.64 ± 0.12, and 0.45 ± 0.07 in folate-low, B(12)-low, and folate/B(12)-low cells, respectively. By contrast, folate and folate/B(12) supplementation resulted in 3.38 ± 0.70 and 2.58 ± 0.14-fold increases in infected macrophages. Interestingly, changes in overall macrophage viability were only observed in folate-high, folate/B(12)-high, and folate/B(12)-low media, with 0.80 ± 0.05, 0.82 ± 0.02, and 0.91 ± 0.04-fold changes, respectively. Incubation of Caco-2 intestinal epithelial monolayers with supernatant from infected macrophages revealed that folate/B(12) deficiency led to increased LDH release independent of oxidative stress. Overall, our results indicate that folate and B(12) are key vitamins affecting cell survival and inflammation during MAP infection.