Research Projects

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), result in approximately 1.5 million deaths every year globally. Mtb is evolved with plethora of host immune evasion strategies such as inhibition of autophagy, host antimicrobial peptides, and induce drug-efflux pumps. It is well understood that Mtb persist in dormancy called “latent tuberculosis” despite of strong host immune responses. However, the questions pertinent to the circumstances under which Mtb is retained in dormancy and the mechanisms of resuscitation to active TB remain important but unresolved. Studies including ours demonstrated that Mtb infect bone marrow mesenchymal stem cells (BM-MSCs) and use them as a safe niche to remain in dormant state by tune-down immune surveillance mechanism(s) of BM cells. Viable Mtb was detected in BM cells who had successfully completed months of anti-Mtb drug treatment and in latently infected TB patients. We showed that Mtb survives in mice Sca-1+/CD44+ BM-MSCs by down-regulating cathelicidin peptide synthesis through TLR2/4 and p38 MAPK pathway. Mtb recruit MSCs at the site of infection to suppress T-cell response. These studies concluded that MSCs serve as a hitherto unappreciated quiescent cellular depot for Mtb. However, underlying mechanisms that tune-down immune surveillance of MSCs to establish Mtb dormancy in BM are not known. Several studies reported that Mtb infection induces different epigenetic modifications in host cells to aid its replication, propagation and protection from the host immune responses. We also found that Mtb promote its survival by inducing H3K9me3 at CIITA promoter to inhibit antigen presentation and autophagy by inducing H3K27me3, and induce host DNA damage by decreasing H3K9me2/3 in macrophages. However, no information is available on the implications of bacterial infections, specifically Mtb on the biology of stem cells. Stem cells express antimicrobial peptides, drug efflux pumps and autophagy that are known to be suppressed by Mtb to aid its survival in macrophages. The proposed study for the first time will undertake comprehensive and systematic epigenome analysis of BM-MSCs during active and dormant stages of Mtb infection. For this, we will employ genome-wide ChIP-Seq and RNA-seq analysis of FACS sorted BM-MSCs during active and dormant Mtb infection. For dormant infection, we have constructed stable MSCs by transfecting Nup98-HoxB4 plasmid. The sequenced ChIP tags from histone marks and RNA Pol-II will be aligned to reference human genome (hg32) using Bowtie2. RNA Pol-II ChIP seq data will be performed using analyzeRNA.pl tool of HOMER suite. Epigenetic modifications will be checked by ChIP assay. The expression of antimicrobial peptides, autophagy, and drug efflux pumps will be checked by Western blot, q-RTPCR and fluorescence microscopy. Ex-vivo results will be validated in in-vivo models by using active TB and latent TB Kramnik mice model developed using C3HeB/FeJ. The global epigenetic changes occurring within Mtb-infected BM-MSCs during both active and dormant infection states are not known. This project will fill this unresolved gap by dissecting the epigenetic landscape during active and dormancy TB, and possibly to identify novel dianostic biomarkes for latent TB. The broad objective of this study is to: “Investigate the global epigenome of BM-MSCs during active and dormant TB infection and to study its implications in the regulation of autophagy, antimicrobial peptides and drug efflux pumps in response to M. tuberculosis infection in bone marrow". These findings may provide basis to understand unresolved fact that BM could be a source of TB reactivation, latency and also possibly emergence of drug-resistant TB. This study is of immense importance for understanding immune dysregulation in TB and offers a novel direction for eliminating dormant Mtb, that may potentially prove to provide more plausible pathways for host directed therapy.