The WGND blog is in the R&D vanguard! Recently, Nick Be wrote a post highlighting the activation of the cellular ‘self-eating’ mechanism known as autophagy in the elimination of TB from an infected individual. On March 5, 2010, Cell published a paper by Kumar and colleagues, which further supports the hypothesis that M. tb infection bypasses host elimination mechanisms, particularly autophagy, in order to survive in host cells, namely macrophages.
Previous investigations on M. tb infection into macrophages have focused on entry and endocytosis, whereas the current study utilized a siRNA approach to identify human host factors (genes within human macrophages) which are increased or decreased to allow M. tb to persist after infection is established. In brief, human macrophage-like cells were plated and 48hrs later infected with M. tb followed by siRNA treatment for 48hrs. Cells were lysed 42hrs later, serial dilutions made, and then dilutions were plated on agar plates to allow bacilli to grow and be counted 18 and 21 days later.
The authors identified 275 host genes through a series of successive experimental procedures which resulted in significant reduction of M. tb load or increased M. tb counts from the original 18, 174 genes represented in the siRNA library. Gene Ontology, a bioinformatics approach, was used to link or cluster the genes into categories of recognized cellular functions. In-depth refinement of the signaling networks obtained revealed sub-networks of gene clusters that were specifically related to the innate and adaptive host immune responses, as well as mediators of inflammatory responses.
To determine whether these apparent 275 pathogen survival genes are common for survival of diverse strains and variants of M. tb, the same siRNA approach used on human macrophage-like cells infected with one of seven independent M. tb field isolates. Results suggest that 74 genes can decrease M. tb load in a strain-independent manner, meaning when these genes are blocked the host cell is able to reduce the amount of bacilli present.
One mechanism to reduce the amount of intracellular M. tb is the activation of ‘self-eating’ or autophagy pathways, as mentioned above. To examine whether autophagy pathways were being activated as a result of the inhibition of these 74 genes, additional experiments were performed with 3-methyladenine (3MA), a classical inhibitor of autophagy. Results revealed that effects of the inhibition of 44 of the 74 genes can be reversed with 3MA, leading to an increase in M. tb in cells.
What does that mean? When M. tb infects cells, it is able to survive and thrive in host cells by regulating the activity of these 44 genes to repress or ‘silence’ host ‘self-eating (autophagy) pathways. Further experiments with siRNA treatment of these 44 genes and interferon gamma (IFN-γ), a cytokine shown to stimulate autophagy, demonstrated a synergistic reduction in M. tb. Overall, data presented suggest that a dual pharmacological approach targeting at least one of the 44 genes involved in repressing host autophagy mechanisms and increasing stimulating cytokines such as IFN-γ could be an effective method of treatment for the elimination of human infection by different strains of M. tb.
Does data presented in this paper suggest a testable alternate approach to target host mechanisms for the elimination of M. tb? Is this approach to targeting host mechanisms ‘safer’ than traditional drug discovery approaches that are targeted towards the pathogen? Assuming some or most of these 44 genes identified are ‘druggable’, what effect, if any, would targeting any of these 44 genes have on other host cells? What are your thoughts and comments?