Folate and Vitamin B12 Deficiency Exacerbate Inflammation during Mycobacterium avium paratuberculosis (MAP) Infection

Abstract

Folate and vitamin B₁₂ 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 (B₉) and cobalamin (B₁₂) deficiency during MAP infection remains uncharacterized. This study investigates how folate and B₁₂ deficiency impedes macrophage apoptosis and exacerbates the inflammation in macrophages infected with MAP isolated from CD patients. Accordingly, we measured folate and B₁₂ 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 B₁₂ deficient media. We determined that MAP-positive CD patients have significantly lower plasma folate and B₁₂ 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 B₁₂ deprivation following MAP infection by several folds, while supplementation significantly reduces their expression by several folds. Additionally, depletion of folate, B₁₂, and folate/B₁₂ 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, B₁₂-low, and folate/B₁₂-low cells, respectively. By contrast, folate and folate/B₁₂ 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/B₁₂-high, and folate/B₁₂-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/B₁₂ deficiency led to increased LDH release independent of oxidative stress. Overall, our results indicate that folate and B₁₂ are key vitamins affecting cell survival and inflammation during MAP infection.

Keywords: B12; B9; Crohn’s disease; MAP; cobalamin; folate; paratuberculosis.

Figure 1

Levels of plasma folate (A) and B₁₂ (B) in MAP-negative and MAP-positive CD patients, N = 35 for both groups. All measurements were performed in duplicates. ** Indicates p-value of less than 0.01.

Figure 2

Effect of folate and B₁₂ deficiency on TNF-α expression in MAP-infected and uninfected macrophages. * Indicates p-value of less than 0.05.

Figure 3

Effect of folate and B₁₂ deficiency on IL-1β expression in MAP-infected and uninfected macrophages. ** Indicates p-value of less than 0.01.

Figure 4

Effect of folate and B₁₂ supplementation on TNF-α expression in MAP-infected and uninfected macrophages. * Indicates p-value of less than 0.05. ** Indicates p-value of less than 0.01.

Figure 5

Effect of folate and B₁₂ supplementation on IL-1β expression in MAP-infected and uninfected macrophages. * Indicates p-value of less than 0.05. ** Indicates a p-value of less than 0.01.

Figure 6

Effect of folate and B₁₂ supplementation or depletion on macrophage apoptosis (A) and general viability (B) during MAP infection. * Indicates p-value of less than 0.05. ** Indicates p-value of less than 0.01.

Figure 7

Effect of folate and vitamin B₁₂ concentration on LDH release from Caco-2 monolayers after treatment with supernatant from infected macrophages. * Indicates p-value less than 0.05. ** Indicates p-value less than 0.01.