Crohn's disease and Mycobacterium avium subsp. paratuberculosis: the need for a study is long overdue

Abstract

The initial suggestion that Mycobacterium avium subsp. paratuberculosis (Map) might be involved in the pathogenesis of Crohn's disease (CD) was based on the apparent similarity of lesions in the intestine of patients with CD with those present in cattle infected with Map, the etiological agent of Johne's disease (JD). Recent investigations have now revealed the presence of Map or Map DNA in blood or lesions from adults and children with CD. Of special interest, Map has also been found in patients with other diseases as well as healthy subjects. The latter observations indicate all humans are susceptible to infection with Map and that, like with other mycobacterial pathogens such as Mycobacterium tuberculosis, infection does not invariably lead to development of clinical disease but rather development of a persistent latent stage of infection where an immune response controls but does not eliminate the pathogen. Limited information has been obtained on the immune response to Map in healthy subjects and patients with CD. Understanding how Map may be involved in the pathogenesis of CD will require a better understanding of the immune response to Map in one of its common hosts as well as healthy humans and patients with CD.

Figure 1

Pictures showing the modified cannula used in the studies (A). Picture of cannula 8 months after surgery in an animal experimentally infected with Map (B). Close up picture showing the condition of the exterior of the cannula 8 months after surgery (C). Close up picture showing the condition of ileal mucosa 8 months following infection with Map (D). Endoscopic field taken before necropsy showing no inflammation in the ileal mucosa (E). Endoscopic field taken before necropsy of a naturally infected animal showing the characteristic swelling and corrugation of the ileal mucosa that occurs at the clinical stage of infection (F). Modified from (Allen et al., 2009; Allen et al., 2011).

Figure 2

Comparison of the expression of CD25 on CD4 and CD8 memory cells stimulated with soluble antigen extract from Map (SAg). Peripheral blood mononuclear cells were collected at the times indicated and stimulated with SAg for 6 days and then processed for flow cytometric analysis (Allen et al., 2009). Electronic gates were set to isolate CD4 and CD8 cells for analysis. The first bar shows pre-inoculation samples that were taken from all of the calves. The second bar shows the mean of the control negative calf sampled over 3 time points and bars 3-5 show the means of the inoculated calves at 4, 5 and 11 months PI. Bar 6 shows expression of CD25 on cells from clinical cows was similar to the expression on cells from experimentally inoculated calves. Asterisks indicate a statistically significance difference P < .05 compared to pre inoculation results. Results from clinical cows were not significantly different from results from calves 11 months PI. From (Allen et al., 2011).

Figure 3

Cytokine gene expression measured by qRT-PCR in lymphocytes from ileocecal lymph nodes from clinical naturally infected cows (CI, n = 3) and experimentally inoculated calves (EI, n = 2) compared to negative controls (NC, n = 3). Control negative values were set at 0. IFN-γ and IL-22 are statistically significant with a P < .05. RQ, relative quantification. From (Allen et al., 2011).

Figure 4

Cytokine gene expression measured by qRT-PCR in PBMCs from control calves (n = 3) and calves experimentally infected with a relA deletion mutant (n = 4) following stimulation with live Map. PBMCs isolated at pre-infection and necropsy were stimulated with live Map for 3 days. The relative transcription was calculated using the value at pre-infection as the calibrator with two housekeeping genes (β-actin and GAPDH). Data are presented as the mean value of each group with error bar (SD). RQ, relative quantification; *, significant difference compared to the value at pre-infection (P < 0.05).

Figure 5

Flow cytometric dot plot profiles comparing the expression of Foxp3 in CD4 T cells from a cow at the pre-clinical stage of infection with CD4 T cells from a cow at the clinical stage of infection. Peripheral blood mononuclear cells from the cows were stimulated with SAg from Map for 6 days. The cells were labeled, with mAbs specific for CD4 (ILA11A, IgG2a), memory T cells (CD45R0, IgG3) then fixed, permeabilized and labeled with a mAb specific for Foxp3 (FOX5A, IgG1) (Seo et al., 2009). Electronic gates were set using side light scatter vs forward light scatter to color code resting, unstimulated cells (red) and activated proliferating cells (blue) (Allen et al., 2009). An additional electronic gate was placed on CD4 T cells (side light scatter vs fluorescence) to isolate CD4 cells for analysis (not shown). As shown in the first profiles (A and B), including the electronic gates containing resting (red) and activated (blue) cells, a subset of activated memory CD4 T cells from the clinical cow expressed Foxp3. In contrast, very few activated cells from the preclinical cow expressed Foxp3. Further analysis of the cells present in the electronic gate only containing activated cells (profiles C and D) revealed a large proportion of the activated cells from the clinical cow expressed Foxp3, showing a potential correlation with disease progression with an increase in CD4 Foxp3 positive T cells.