Role of TNF in the Altered Interaction of Dormant Mycobacterium tuberculosis with Host Macrophages

Abstract

Mycobacterium tuberculosis (Mtb) persists within lung granulomas, despite being subjected to diverse stress conditions, including hypoxia. We hypothesized that the response of host phagocytes to Mtb experiencing hypoxia is radically altered and designed in vitro experiment to study this phenomenon. Hypoxia-stressed (Mtb-H) and aerobically grown Mtb (Mtb-A) were used to infect Rhesus Macaque Bone Marrow Derived Macrophages (Rh-BMDMs) and the comparative host response to Mtb infection studied. Mechanistic insights were gained by employing RNAi. Mtb-H accumulated significantly lower bacterial burden during growth in Rh-BMDMs, concomitantly generating a drastically different host transcriptional profile (with only <2% of all genes perturbed by either infection being shared between the two groups). A key component of this signature was significantly higher TNF and apopotosis in Mtb-H- compared to Mtb-A-infected Rh-BMDMs. Silencing of TNF by RNAi reversed the significant control of Mtb replication. These results indicate a potential mechanism for the rapid clearance of hypoxia-conditioned bacilli by phagocytes. In conclusion, hypoxia-conditioned Mtb undergo significantly different interactions with host macrophages compared to Mtb grown in normoxia. These interactions result in the induction of the TNF signaling pathway, activation of apoptosis, and DNA-damage stress response. Our results show that Mtb-H bacilli are particularly susceptible to killing governed by TNF.

DosS Is Required for the Complete Virulence of Mycobacterium tuberculosis in Mice with Classical Granulomatous Lesions

“…Lung tissue was fixed for hematoxylin-eosin histology and confocal microscopy (18)(19)(20)(21)(22). Ten images of each slide were used for analysis (Inform; PerkinElmer, Waltham, MA).…”

“…For macrophage infection studies, RNA was obtained as described (20), and comparisons were performed between cells infected with Mtb and D-dosS. Data were analyzed as described elsewhere Figure 1A).…”

The Mycobacterium tuberculosis Clp Gene Regulator Is Required for in Vitro Reactivation from Hypoxia-induced Dormancy

“…Data can be retrieved using the GEO platform number GPL18320. For RT-PCR, RNA was treated with DNase as previously described (17,22,24). RNA was reverse-transcribed following the manufacturers' instructions using the High Capacity RNA-to-cDNA kit (Applied Biosystems) (17,22,24).…”

“…For RT-PCR, RNA was treated with DNase as previously described (17,22,24). RNA was reverse-transcribed following the manufacturers' instructions using the High Capacity RNA-to-cDNA kit (Applied Biosystems) (17,22,24). RT PCR was performed as per the manufacturers' instructions using Power SYBR Green PCR Master Mix (Applied Biosystems) and as previously described (17,22,24).…”

ClgR contributes to pulmonary pathology but not bacterial growth in Mycobacterium tuberculosis infection

“…Isolation and maintenance of Rhesus Bone Marrow Derived Macrophages (Rh-BMDMs) as well as their infection with Mtb, isogenic mutant ΔclgR and a complemented strain was carried out strictly as per the experimental details described previously [21]. Briefly, Rh-BMDMs were cultured in IMDM complete media (Gibco) (IMDM media supplemented with 10% heat-inactivated FBS (Hyclone) and 1% Penicillin/Streptomycin mix (Pen/Strep, Gibco).…”