Introduction and Background of the Project

Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a highly successful pathogen with one third of the human population currently infected. Indeed, annually 9.6 million people become ill with TB, and 1.5 million die from the disease worldwide. Unfortunately, current BCG vaccine efficacy is limited and very few new antibiotics are in the pipeline. More seriously, drug resistance of current Mtb drugs is increasing at an alarming rate. Mtb survives in host macrophages by exploiting several cellular host factors. However it is unclear how Mtb modulates the transcriptional landscape of macrophages to establish a persistence infection.

Project objective

The objectives of this project is to identify host genes, signaling pathways and mechanisms hijacked by Mycobacterium tuberculosis (Mtb) that are critical for entry, survival and replication in human macrophages, as host-directed drug targets to combat tuberculosis.

Project design

We have already produced a large scale transcriptome data for tuberculosis infection using mouse bone marrow-derived macrophage cells (BMDM). We continue the detailed data analysis to extract genes of interests. The selected genes will be explored/confirmed by literature profiling and transcriptome analysis of Mtb-infected human monocyte-derived macrophages cells (MDM). The promising genes will be explored to clarify their host detrimental or host protective function during tuberculosis infection. The biological relevance of our priority targets on mycobacterial growth and host protective immune functions will be investigated in Mtb-infected BMDM/MDM using in-house generated lentivirus-mediated shRNA gene knockdown.

How can your partnership (project) address global health challenges?

Our strategy is less likely to engender resistance for the bacteria by adaptation, and supplementing pathogen-directed targeting by antibiotic treatments. This would prolong the life span of antibiotics, thereby reducing the frequency of treatment failures.

What sort of innovation are you bringing in your project?

Our innovations are based on our unique transcriptome technology, deepCAGE (1, 2). We have applied this technology to the macrophage transcriptome project for Mycobacterium tuberculosis (Mtb) infection, where we are particularly interested in how macrophage, the host cell of Mtb, will be transcriptionally altered by Mtb infection. This knowledge will support our efforts for host-directed drug therapy. Hence, we redefined the transcriptional regulatory dynamics of classically and alternatively activated macrophages using our transcriptome technology (3). We uncovered a novel transcription factor Batf2 in macrophage cells and demonstrated that Batf2, together with Irf1, induces inflammatory responses in classically activated macrophages, lipopolysaccharides, as well as  mycobacterial infection (4). We further demonstrated that intracellular Mtb drives the macrophage activation to an alternative status by activating pathways, downstream from IL-4-mediated activation as an evasion mechanism (5). Those achievements, among other highly interesting gene products, allows us now to interfere by host-directed targeting to reverse Mtb evasion (6). Moreover, we also demonstrated that Mtb exploits the host cholesterol pathway for its survival and were able to therapeutically increase protection against tuberculosis by reducing cholesterol by statins. This resulted in cholesterol phagosomal maturation of the host and increased macrophage autophagy, both important to kill Mtb intracellularly (7). These results demonstrated the proof of principle for host-directed drug targeting (8) with several interesting genes in our pipeline to further explore their gene products in host protection or pathogen evasion.

Role and Responsibility of Each Partner

1. The designated development partner: RIKEN Center for Life Science Technologies

・Analysis of large scale transcriptome data in Mtb-infected BMDM.

・Transcriptome data production of human Mtb-infected MDM.

・Analysis of Mtb-infected MDM.

・Creation of lentivirus for gene knockdown studies.

2. The collaborating partner: International Centre for Genetic Engineering and Biotechnology (ICGEB)

・Analysis of large scale transcriptome data in Mtb-infected BMDM.

・Analysis of Mtb-infected MDM.

・In vitro evaluation of host genes affecting tuberculosis infection and growth using lentivirus-mediated gene knockdown.

Others (including references if necessary)

1. Arner, E., Daub, C.O., Vitting-Seerup, K., Andersson, R., Lilje, B., Drablos, F., Lennartsson, A., Ronnerblad, M., Hrydziuszko, O., Vitezic, M., et al. 2015. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells. Science 347:1010-1014.

2. Forrest, A.R., Kawaji, H., Rehli, M., Baillie, J.K., de Hoon, M.J., Haberle, V., Lassman, T., Kulakovskiy, I.V., Lizio, M., Itoh, M., et al. 2014. A promoter-level mammalian expression atlas. Nature 507:462-470.

3. Roy, S., Schmeier, S., Arner, E., Alam, T., Parihar, S.P., Ozturk, M., Tamgue, O., Kawaji, H., de Hoon, M.J., Itoh, M., et al. 2015. Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics. Nucleic Acids Res 43:6969-6982.

4. Roy, S., Guler, R., Parihar, S.P., Schmeier, S., Kaczkowski, B., Nishimura, H., Shin, J.W., Negishi, Y., Ozturk, M., Hurdayal, R., et al. 2015. Batf2/Irf1 induces inflammatory responses in classically activated macrophages, lipopolysaccharides, and mycobacterial infection. J Immunol 194:6035-6044.

5. Guler, R., Parihar, S.P., Savvi, S., Logan, E., Schwegmann, A., Roy, S., Nieuwenhuizen, N.E., Ozturk, M., Schmeier, S., Suzuki, H., et al. 2015. IL-4Ralpha-dependent alternative activation of macrophages is not decisive for Mycobacterium tuberculosis pathology and bacterial burden in mice. PLoS One 10:e0121070.

6. Guler, R., Roy, S., Suzuki, H., and Brombacher, F. 2015. Targeting Batf2 for infectious diseases and cancer. Oncotarget 6:26575-26582.

7. Parihar, S.P., Guler, R., Khutlang, R., Lang, D.M., Hurdayal, R., Mhlanga, M.M., Suzuki, H., Marais, A.D., and Brombacher, F. 2014. Statin therapy reduces the mycobacterium tuberculosis burden in human macrophages and in mice by enhancing autophagy and phagosome maturation. J Infect Dis 209:754-763.

8. Guler, R., and Brombacher, F. 2015. Host-directed drug therapy for tuberculosis. Nat Chem Biol 11:748-751.

Final Report

1. Project objective

The objectives of this project is to identify host genes, signaling pathways and mechanisms hijacked by Mycobacterium tuberculosis (Mtb) that are critical for entry, survival and replication in human macrophages, for host-directed drug targets. Ninety percent of latent infected people, who stay clinically immune to the disease for their life, must have effective host protective immune responses. Exploring and taking advantage of these efficient host-protected defense mechanisms, the topic of this application is to identify host-directed drug targets to augment host immunity and counteract host factors hijacked by the pathogen, as part of its evasion mechanism.

2. Project design

We take advantage of deepCAGE, our unique promoter-based transcriptome technology. We have already produced mouse bone marrow-derived macrophages (BMDM) time course transcriptome data of Mtb-infection under classical and alternative activation. We first analyze differentially expressed genes, either Mtb-infection-specific and/or dependent on the activation state of the infected macrophages, to identify host genes considered to be involved in Mtb-infection, survival and growth. Next, those genes will be evaluated by our already established assay system using lentivirus gene perturbation system. The promising genes will be further evaluated using monocyte-derived macrophage cells in human and using gene KO mice.

3. Results, lessons learned

Using our published Mtb-infected mouse BMDM transcriptome data (Sci Rep. 8, 6758, 2018), we extracted drastically up- and down-regulated protein coding host genes during infection. We further created Mtb-infected human monocyte-derived macrophages (MDM) transcriptome data using the deepCAGE technology. We have established the method to select validation targets and ranked selected targets accordingly. For our priority targets, we have selected CMPK2 and RSAD2 for functional evaluation. We have created 5 shRNA constructs per gene and examined the knockdown effect in Mtb-infected MOUSE BMDM. Results showed that RSAD2 and CMPK2 knockdowns moderately decreased the intracellular growth of Mtb in macrophages. In the gene expression analysis of active TB patients, we found that their mRNA expression level was drastically higher in the TB patients when compared to healthy controls. We have also examined DAXX2 and CXCL4 as targets where specific inhibitors are available, and IGF1 for those with repurposed drugs availability. DAXX2 inhibitor, berberine, showed decrease in Mtb growth in both HUMAN and MOUSE macrophages without affecting cell viability. Furthermore, berberine-treated mice displayed reduced inflammation with decreasing proinflammatory cytokines and immune cell populations in the lungs without affecting the bacterial burdens. Berberine in combination with rifampin/isoniazid increased their efficacy in reducing inflammation, proinflammatory cytokines and immune cell populations in the lungs. Furthermore, berberine increased the microbicidal activity of rifampin/isoniazid in the spleen. Therefore, berberine as an adjunct agent has the potential for host-directed therapy against tuberculosis to reduce pathology and mycobacterial dissemination. CXCL4 was neutralized using PF4 antibody, macrophages neutralized with anti-PF4 antibody displayed decreased growth of Mtb at later time point of infection. IGF-1 was shown to decreased Mtb growth upon lentivirus knockdown and inhibitor (tyrphostin). We used a more specific inhibitor picropodophyllin (PPP) of IGF-1 and found a more effective inhibition in the intracellular growth of Mtb in HUMAN macrophages as opposed to MOUSE macrophages. We will further examine whether PPP could be used as an adjunct to standard anti-tubercular therapy. In conclusion, the host-directed drug targets against tuberculosis, shown above, are promising, and we are planning to apply to the next GHIT grant application for their further functional analysis.