Involvement of mycobacterium avium subspecies paratuberculosis in TNF-α production from macrophage: possible link between MAP and immune response in Crohn's disease.

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. We note with great interest ‘‘Mycobacterium avium subsp. paratuberculosis (MAP) as a Modifying Factor in Crohn’s Disease’’ by Sibartie et al. 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)a 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. 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 MAPinfected 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). Next, we investigated cytokine production from THP-1 cells infected with viable MAP and MAV. THP-1 cells were coincubated with MAP or