To the Editor:

Crohn's disease (CD) is a chronic disease characterized by recurrent and severe inflammation of the gastrointestinal tract. Despite the unknown etiology, it is considered that chronic activation of the innate and adaptive mucosal immune systems in a genetically susceptible host results in the development of CD and that enteric microflora play a central role in initiation and maintenance of disease.

Mycobacterium avium subspecies paratuberculosis (MAP) has the specific ability to cause chronic bowel inflammation with histopathological features of granulomatous enteritis in ruminants. Systematic and meta-analysis of research have shown the relationship between MAP infection and CD, although the evidence remains inconclusive.1

We note with great interest “Mycobacterium avium subsp. paratuberculosis (MAP) as a Modifying Factor in Crohn's Disease” by Sibartie et al.2 The authors examined the influence of MAP on T-cell proliferation and cytokine response in patients with inflammatory bowel disease (IBD). As a result, coincubation of peripheral blood mononuclear cells (PBMCs) with MAP induced significantly more T-cell proliferation in PBMCs isolated from CD patients compared to those with ulcerative colitis (UC) or healthy volunteers. Also, PBMCs from CD patients produced significantly higher levels of tumor necrosis factor (TNF)-α in response to MAP compared to UC and controls. They concluded that these data did not support a causal effect for MAP but altered immune reactivity to microbes and their components in CD. However, we still have the following question: Which is important to induce several cytokine productions from immune cells, viable MAP or components of MAP? Or what cytokine is mainly induced from macrophages in which viable MAP could exist? A recent study has demonstrated an association in IBD with a coding variant of ATG16L, IL-23R, and IRGM genes, thereby implicating the autophagy pathway that is crucial in inhibiting M. tuberculosis survival in infected macrophages.3, 4 However, several reports suggested that it seemed to be difficult to detect viable MAP by isolation of the organism using culture methods and the effect of viable MAP on cytokine production from macrophages remains unclear. In this regard, we herein show data on the infectivity of MAP in a human monocyte cell line, THP-1 cells, and the cytokine production from MAP-infected cells.

A human monocyte cell line, THP-1 cells, were stimulated with 16 nM of phorbol 12-myristate 13-acetate and then coincubated with mycobactriums for 4 days and serial change of the number of intracellular bacteria were investigated by colony-forming unit (CFU) assay. First, Ziehl-Neelsen staining showed the existence of viable MAP in stimulated THP-1 cells (Fig. 1). The number of viable MAP in THP-1 cells were significantly more than those in Mycobacterium smegmatis, which has been confirmed to have no pathogenicity for humans. On the contrary, there was no significant difference of the number between MAP and M. avium (MAV), which is a causative microorganism of human atypical mycobacteriosis (Fig. 2).

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Figure 1. Microscopic examination of viable MAP by acid-fast Ziehl-Neelsen staining (arrows). [Color figure can be viewed in the online issue, which is available at]

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Figure 2. Serial change of the number of intracellular mycobacteria (CFU assay). [Color figure can be viewed in the online issue, which is available at]

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Next, we investigated cytokine production from THP-1 cells infected with viable MAP and MAV. THP-1 cells were coincubated with MAP or MAV for 12 hours. After eradication of extracellular bacteria, concentrations of TNF-α, IL-12p40, and IL-6 in the supernatant of culture medium were determined by enzyme-linked immunosorbent assay (ELISA). TNF-α production was significantly higher from viable MAP-infected THP-1 cells than MAV-infected cells, while it did not induce IL-12p40 or IL-6 (Fig. 3).

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Figure 3. Cytokine production for THP-1 cells stimulated with mycobacteria. [Color figure can be viewed in the online issue, which is available at]

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This study demonstrates that MAP-infected THP-1 cells as well as MAV and production of TNF-α was significantly higher in viable MAP-infected THP-1 cells compared to that from MAV-infected cells despite no difference of intracellular number of viable bacteria between them. Most important, our data clearly showed that viable MAP in THP-1 cells induced cytokine production, particularly TNF-α, because previous studies did not reveal any proof that viable MAP could affect cytokine production from macrophages despite showing cytokine production from immune cells coincubated with MAP.2 It was reported that significantly higher TNF-α concentrations were found in culture supernatants for CD compared to UC, irritable bowel syndrome, or controls and level of TNF-α were correlated with the presence of MAP.5 Taken together, a higher level of TNF-α production in response to MAP may be involved in patients with CD who have a potent response to anti-TNF therapies.

Next, we found that THP-1 cells infected with MAP did not produce IL-12p40 compared to those with MAV. These data suggest that MAP is not involved in cytokine production linked to Th1 and Th17, which are characteristic immune responses of CD. IL-12 was previously shown to be essential in host response to mycobacteria in human patients because it is known to be a major inducer of IFN-γ production by T and NK cells; IFN-γ induces macrophages to activate their bactericidal activities. In this regard, high production of TNF-α from macrophages infected with viable MAP might contribute to the pathogenesis of CD, while lack of IL-12 production could help MAP live in macrophages. In accordance with our results, Campos et al6 also reported that MAP failed to elicit IL-12 secretion by macrophages from healthy controls, at any infection timepoint tested. These results may support that viable MAP does not affect activation of IRF-8 as a major transcriptional factor regulating IL-12p40, although the exact reason why MAP did not affect IL-12 production from macrophages should be elucidated in the future.

Interestingly, our data also demonstrated that MAP did not elicit IL-6 production from THP-1 cells, while MAV could induce it. Previously, we showed no significant association between the serum level of anti-IS900 and disease activity of CD, suggesting that MAP may not always elicit strong immune responses in the human intestine even if it is infected, but interfere with local immune response.7 In conclusion, MAP might be a specific bacterium for direct induction of TNF-α alone, and involved in the pathophysiology of patients with CD positive for MAP.


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  • 1
    Feller M, Huwiler K, Stephan R, et al. Mycobacterium avium subspecies paratuberculosis and Crohn's disease: a systematic review and meta-analysis. Lancet Infect Dis. 2007; 7: 607613.
  • 2
    Sibartie S, Scully P, Keohane J, et al. Mycobacterium avium subsp. Paratuberculosis (MAP) as a modifying factor in Crohn's disease. Inflamm Bowel Dis. 2010; 16: 296304.
  • 3
    Duerr RH, Taylor KD, Brant SR, et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science. 2006; 314: 14611463.
  • 4
    Parkes M, Barrett JC, Prescott NJ, et al. Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn's disease susceptibility. Nat Genet. 2007; 39: 830832.
  • 5
    Clancy R, Ren Z, Turton J, et al. Molecular evidence for Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn's disease correlates with enhanced TNF-alpha secretion. Dig Liver Dis. 2007; 39: 445451.
  • 6
    Campos N, Magro F, Castro AR, et al. Macrophages from IBD patients exhibit defective tumour necrosis factor-α secretion but otherwise normal or augmented pro-inflammatory responses to infection. Immunobiology. 2011 [Epub ahead of print].
  • 7
    Nakase H, Nishio A, Tamaki H, et al. Specific antibodies against recombinant protein of insertion element 900 of Mycobacterium avium subspecies paratuberculosis in Japanese patients with Crohn's disease. Inflamm Bowel Dis. 2006; 12: 6269.

Hiroshi Nakase MD, PhD*, Hiroyuki Tamaki MD, PhD*, Minoru Matsuura MD, PhD*, Tsutomu Chiba MD, PhD*, Kazuichi Okazaki MD, PhD†, * Department of Gastroenterology and Hepatology, Graduate School of Medicine Kyoto University, Kyoto, Japan, † Third Department of Internal Medicine Division of Gastroenterology and Hepatology, Kansai Medical University Kansai, Japan.