2'-5'-Oligoadenylate synthetase-like protein inhibits intracellular M. tuberculosis replication and promotes proinflammatory cytokine secretion.

Host cytoplasmic surveillance pathways are known to elicit type I interferon (IFN) responses which are crucial to antimicrobial defense mechanisms. Oligoadenylate synthetase-like (OASL) protein has been extensively characterized as a part of the anti-viral mechanism, however a number of transcriptomic studies reveal its upregulation in response to infection with a wide variety of intracellular bacterial pathogens. To date, there is no evidence documenting the role (if any) of OASL during mycobacterium tuberculosis infection. Using two pathogenic strains differing in virulence only, as well as the non-pathogenic M. bovis BCG strain, we observed that pathogenicity and virulence strongly induced OASL expression after 24 h of infection. Further, we observed that OASL knock down led to a significant increase in M. tb CFU counts 96 h post-infection in comparison to the respective controls. Luminex revealed that OASL silencing significantly decreased IL-1ß, TNF-α and MCP-1 secretion in THP-1 cells and had no effect on IL-10 secretion. We therefore postulate that OASL regulates pro-inflammatory mediators such as cytokines and chemokines which suppress intracellular mycobacterial growth and survival.

Comparison of human monocyte derived macrophages and THP1-like macrophages as in vitro models for M. tuberculosis infection.

Macrophages are the preferential cell types to study various aspects of mycobacterial infection. Commonly used infection models for in-vitro studies are primary macrophages such as human monocyte derived macrophages (hMDMs) and macrophage like cell lines (THP-1). It is not clear if commercially available THP-1 cells can be used as hMDMs alternative for in-vitro M.tb infection experiments. We conducted a detailed investigation of the hMDM and THP-1 response to mycobacterial infection on a comparative basis and assess the most crucial aspects of infection which are most commonly studied. We assessed mycobacterial uptake and intracellular growth over time of a pathogenic drug-resistant and drug-susceptible M.tb strains (R179 and H37Rv) through colony forming units (CFUs). Both strains depicted similar uptake and intracellular growth in hMDMs and THP-1 macrophages over time (R179, p = 0.954) (H37Rv, p = 0.922). Cytotoxicity assays revealed a consistent viability up to day 16 post-infection across the strains in both THP-1 and hMDMs (R179, p = 0.271) (H37Rv, p = 0.068). Interestingly, both cell lines showed similar mycobacterial uptake and cellular viability in both susceptible as well as resistant M.tb strains. Cytokine/chemokine mRNA analysis through qPCR found no difference between cell types. Further, cytokine secretion measured through Luminex revealed no difference across the strains. Also, cytokine secretion analysis showed no difference in both cell lines across strains. In conclusion, our study shows that THP-1 and hMDMs bacterial uptake, viability and host response to drug-susceptible and drug-resistant mycobacterial infections are similar. Therefore, present study demonstrate that THP-1 cells are suitable substitutes for hMDMs for in-vitro M.tb infection experiments.

Virulence, biochemistry, morphology and host-interacting properties of detergent-free cultured mycobacteria: An update.

The culturing of mycobacteria is a standard procedure that is consistent world-wide, with little variation in the growth media constituents, particularly those found in liquid and solid media. Before the 1940s however, the aggregating nature of mycobacteria as well as the characteristic slow growth-rate saw mycobacterial research delay considerably. Dubos and colleagues addressed both these issues and observed that a very small volume of Tween detergent was sufficient to greatly improve the culturing of mycobacteria. Over the years however, evidence of the unfavourable effects of this detergent on a number of morphological, biochemical, pathogenic and host-interacting properties of mycobacteria surfaced. For the first time we bring together literature, past and present to comprehensively review the mycobacterial properties which are, and are not affected by the use of this detergent. We also address other detergents and methods which may circumvent the need to include Tween compounds in mycobacterial culture media.

The role of mTOR during cisplatin treatment in an in vitro and ex vivo model of cervical cancer.

Cisplatin is used as a cytotoxic agent for the management of cervical cancer. However, the severity of the side-effects limits the use of this drug, particularly at high doses. Resistance to cisplatin is often attributed to a disruption in the normal apoptotic response via aberrant activation of pathways such as the mTOR pathway. Here we assess the role of mTOR and its effect on cell death sensitization and autophagy in response to a low concentration of cisplatin in cervical cancer cells. Additionally we measured the expression profile of mTOR in normal, low- and high-grade squamous intraepithelial (LSIL and HSIL) lesions and cancerous tissue. An in vitro model of cervical cancer was established using HeLa and CaSki cells. mTOR protein expression as well as autophagy-related proteins were evaluated through Western blotting. Inhibition of mTOR was achieved with the use of rapamycin and RNA silencing. A low concentration of cisplatin administered as a single agent induces autophagy, but not apoptosis. Cisplatin cytotoxicity was greatly enhanced in cancer cells when mTOR had been inhibited prior to cisplatin treatment which was likely due to autophagy being increased above cisplatin-induced levels, thereby inducing apoptosis. Cervical tissue samples revealed an increase in mTOR protein expression in LSIL and carcinoma tissue which suggests a change in autophagy control. Our data suggest that utilising a lower dose of cisplatin combined with mTOR inhibition is a viable treatment option and addresses the challenge of cisplatin dose-dependent toxicity, however future studies are required to confirm this in a clinical setting.