Microscopy, cytometry help make important discovery about tuberculosis

Researchers at the Forsyth Institute (Cambridge, MA) used confocal microscopy and flow cytometry to help study latent tuberculosis (TB) and the bacteria's ability to hide in stem cells.

Related: DNA sequencing can trace the spread of drug-resistant tuberculosis

Some bone marrow stem cells reside in low oxygen (hypoxia) zones, which immune cells and toxic chemicals cannot reach. Hypoxia-activated cell signaling pathways may also protect the stem cells from dying or aging. A new study, led by Bikul Das, MBBS, Ph.D., associate research investigator at the Forsyth Institute and the honorary director of the KaviKrishna laboratory (Guwahati, India), has found that Mycobacterium tuberculosis (Mtb) hijack this protective hypoxic zone to hide intracellularly to a special stem cell type.

Earlier research at Forsyth revealed that Mtb hides inside a specific stem cell population in bone marrow, the CD271+ mesenchymal stem cells. However, the exact location of the Mtb harboring stem cells was not known.

Das and his collaborators at Jawarharlal Nehru Univeristy (JNU; New Delhi, India) and KaviKrishna Laboratory used a well-known mouse model of Mtb infection, where months after drug treatment, Mtb remain dormant for future reactivation. Using this mouse model of dormancy, scientists isolated the special bone marrow stem cell type, the CD271+ mesenchymal stem cells, from the drug-treated mice. Prior to isolation of the stem cells, mice were injected with pimonidazole, a chemical that binds specifically to hypoxic cells. Pimonidazole binding of these cells was visualized under a confocal microscope and via flow cytometry. The scientists found that despite months of drug treatment, Mtb could be recovered from the CD271+ stem cells. Most importantly, these stem cells exhibit strong binding to pimonidazole, indicating the hypoxic localization of the stem cells. Experiments also confirmed that these stem cells express a hypoxia-activated gene, the hypoxia-inducible factor 1 alpha (HIF-1alpha).

To confirm the findings in clinical subjects, the research team isolated the CD271+ stem cell type from the bone marrow of TB-infected human subjects who had undergone extensive treatment for the disease. They found that not only did the stem cell type contain viable Mtb, but also exhibit strong expression of HIF-1alpha. To their surprise, the CD271+ stem cell population expressed several-fold higher expression of HIF-1alpha than the stem cell type obtained from the healthy individuals.

"These findings now explain why it is difficult to develop vaccines against tuberculosis," Das says. "The immune cells activated by the vaccine agent may not be able to reach the hypoxic site of bone marrow to target these 'wolfs-in-stem-cell-clothing.'"

Now, the research team plans to develop a Forsyth Institute/KaviKrishna Laboratory global health research initiative to advance stem cell research and its application to global health issues, including TB, HIV, and oral cancer, all critical problems in the area where KaviKrishna Laboratory is located.

Full details of the work appear in the American Journal of Pathology; for more information, please visit http://dx.doi.org/10.1016/j.ajpath.2015.03.028.

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