Mycobacterium tuberculosis Induces Irg1 in Murine Macrophages by a Pathway Involving Both TLR-2 and STING/IFNAR Signaling and Requiring Bacterial Phagocytosis.

Irg1 is an enzyme that generates itaconate, a metabolite that plays a key role in the regulation of inflammatory responses. Previous studies have implicated Irg1 as an important mediator in preventing excessive inflammation and tissue damage in Mycobacterium tuberculosis (Mtb) infection. Here, we investigated the pattern recognition receptors and signaling pathways by which Mtb triggers Irg1 gene expression by comparing the responses of control and genetically deficient BMDMs. Using this approach, we demonstrated partial roles for TLR-2 (but not TLR-4 or -9), MyD88 and NFκB signaling in Irg1 induction by Mtb bacilli. In addition, drug inhibition studies revealed major requirements for phagocytosis and endosomal acidification in Irg1 expression triggered by Mtb but not LPS or PAM3CSK4. Importantly, the Mtb-induced Irg1 response was highly dependent on the presence of the bacterial ESX-1 secretion system, as well as host STING and Type I IFN receptor (IFNAR) signaling with Type II IFN (IFN-γ) signaling playing only a minimal role. Based on these findings we hypothesize that Mtb induces Irg1 expression in macrophages via the combination of two independent triggers both dependent on bacterial phagocytosis: 1) a major signal stimulated by phagocytized Mtb products released by an ESX-1-dependent mechanism into the cytosol where they activate the STING pathway leading to Type I-IFN production, and 2) a secondary TLR-2, MyD88 and NFκB dependent signal that enhances Irg1 production independently of Type I IFN induction.

Combining Host Genetics and Functional Analysis to Depict Inflammasome Contribution in Tuberculosis Susceptibility and Outcome in Endemic Areas.

The interplay between M. tuberculosis (Mtb) and humans is multifactorial. The susceptibility/resistance profile and the establishment of clinical tuberculosis (TB) still remains elusive. The gain-of-function variant rs10754558 in the NLRP3 gene (found in 30% of the world population) confers protection against the development of TB, indicating a prominent role played by NLRP3 inflammasome against Mtb. Through genotype-guided assays and various Mtb strains (BCG, H37Rv, Beijing-1471, MP287/03), we demonstrate that Mtb strains activate inflammasome according to the NLRP3/IL-1ß or NLRC4/IL18 preferential axis. NLRP3 and NLRC4 genetic variants contribute to the presentation of TB. For the first time, we have shown that loss-of-function variants in NLRC4 significantly contribute to the development of extra-pulmonary TB. The analysis of inflammasome activation in a cohort of TB patients and their "household contacts" (CNT) revealed that plasma IL-1ß/IFN-α ratio lets us distinguish patients from Mtb-exposed-but-healthy individuals from an endemic region. Moreover, NLRP3 inflammasome seemed "exhausted" in TB patients compared to CNT, indicating a more efficient activation of inflammasome in resistant individuals. These findings suggest that inflammasome genetics as well as virulence-dependent level of inflammasome activation contribute to the onset of a susceptible/resistant profile among Mtb-exposed individuals.

Synthesis, Cytotoxic Activity on Leukemia Cell Lines and Quantitative Structure-Activity Relationships (QSAR) Studies of Morita-Baylis-Hillman Adducts.

BACKGROUND: The Morita-Baylis-Hillman reaction is an organocatalyzed chemical transformation that allows access to small poly-functionalized molecules and has considerable synthetic potential and promising biological profiles. The Morita-Baylis-Hillman adducts (MBHA) are a new class of bioactive compounds and highlight its potentialities to the discovery of new cheaper and efficient drugs, e.g. as anti-Leishmania chagasi and Leishmania amazonensis, anti- Trypanosoma cruzi, anti-Plasmodium falciparum and Plasmodium berghei, lethal against Biomphalaria glabrata, antibacterial, antifungal, herbicide and others. METHODS: The goal of this work is to describe the primary cytotoxic activities against strains of human leukemia HL-60 cell line for thirty-four Morita-Baylis- Hillman adducts (MBHA), followed by a Quantitative Structure-Activity Relationships study (QSAR). RESULTS: The conventional or microwave-assisted syntheses of MBHA, derived from substituted aromatics or Isatin, were performed in good to excellent yields (70-100%) in short reaction times, using protocols recently developed by us. Isatin derivatives, MBHA 31 and 32, were the most active in this congener series of compounds, with IC50 values of 10.8 µM and 7.8 µM, respectively. The primary cytotoxic activities against chronic leukemia cells (K562) were also evaluated to these two most active compounds (MBHA 31 and 32), presenting IC50 values of 53 µM and 43 µM respectively. QSAR study was performed considering 3D, 2D and constitutional molecular descriptors. These were selected from Ordered Predictor Selection algorithm and submitted to Partial Least Squares Modeling. CONCLUSION: We present an interesting investigation about cytotoxic activities on human leukemia cell line (HL-60) for 34 synthetic MBHA. In a good way we discovered that the most cytotoxic compounds (31-32, 10.8 µM and 7.8 µM respectively) were also prepared quantitatively (100% yields) in a short reaction time using microwave irradiation. We demonstrate that 31 and 32 induced apoptosis and not necrosis in HL-60 cells, observed by externalization of PS and increase Anexin-V positive cells. Quantitative Structure-Activity Relationships considering 3D, 2D and constitutional descriptors provided a robust and predictive PLS model, in accordance with SAR observations.