Mycobacterium tuberculosis (Mtb) extracellular DNA (eDNA) gains access to the host cell cytosol via the ESX-1 secretion system. It is puzzling that this eDNA of Mtb does not induce activation of the AIM2-inflammasome since AIM2 recognizes cytosolic DNA. Here we show that non-virulent mycobacteria such as M. smegmatis induce AIM2-inflammasome activation, which is dependent upon their strong induction of IFN-β production. In contrast, Mtb, but not an ESX-1 deficient mutant, inhibits the AIM2-inflammasome activation induced by either M. smegmatis or transfected dsDNA. The inhibition does not involve changes in host cell AIM2 mRNA or protein levels but led to decreased activation of caspase-1. We furthermore demonstrate that Mtb inhibits IFN-β production and signaling, which was partially responsible for the inhibition of AIM2 activation. In conclusion, we report a novel immune evasion mechanism of Mtb that involves the ESX-1-dependent, direct or indirect, suppression of the host cell AIM2-inflammasome activation during infection.

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Background Interleukin-1β (IL-1β) is important for host resistance against Mycobacterium tuberculosis (Mtb) infections. The response of the dendritic cell inflammasome during Mtb infections has not been investigated in detail. Methodology/Principal Findings Here we show that Mtb infection of bone marrow-derived dendritic cells (BMDCs) induces IL-1β secretion and that this induction is dependent upon the presence of functional ASC and NLRP3 but not NLRC4 or NOD2. The analysis of cell death induction in BMDCs derived from these knock-out mice revealed the important induction of host cell apoptosis but not necrosis, pyroptosis or pyronecrosis. Furthermore, NLRP3 inflammasome activation and apoptosis induction were both reduced in BMDCs infected with the esxA deletion mutant of Mtb demonstrating the importance of a functional ESX-1 secretion system. Surprisingly, caspase-1/11-deficient BMDCs still secreted residual levels of IL-1βand IL-18 upon Mtb infection which was abolished in cells infected with the esxA Mtb mutant. Conclusion Altogether we demonstrate the partially caspase-1/11-independent, but NLRP3- and ASC- dependent IL-1β secretion in Mtb-infected BMDCs. These findings point towards a potential role of DCs in the host innate immune response to mycobacterial infections via their capacity to induce IL-1β and IL-18 secretion.

Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to, or directly target lysosomes, the major intracellular degradative organelle. Here, we demonstrate that AGS3 (Activator of G-protein Signaling 3), a lipopolysaccharide inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GPR/GoLoco) motif stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin (mTOR) pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB (TFEB), a known activator of lysosomal gene transcription. In contrast, AGS3 deficient macrophages had increased mTOR activity, reduced TFEB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus while AGS3 deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

The interaction of Mycobacterium tuberculosis (Mtb) with host cell death signaling pathways is characterized by an initial anti-apoptotic phase followed by a pro-necrotic phase to allow for host cell exit of the bacteria. The bacterial modulators regulating necrosis induction are poorly understood. Here we describe the identification of a transcriptional repressor, Rv3167c responsible for regulating the escape of Mtb from the phagosome. Increased cytosolic localization of MtbΔRv3167c was accompanied by elevated levels of mitochondrial reactive oxygen species and reduced activation of the protein kinase Akt, and these events were critical for the induction of host cell necrosis and macroautophagy. The increase in necrosis led to an increase in bacterial virulence as reflected in higher bacterial burden and reduced survival of mice infected with MtbΔRv3167c. The regulon of Rv3167c thus contains the bacterial mediators involved in escape from the phagosome and host cell necrosis induction, both of which are crucial steps in the intracellular lifecycle and virulence of Mtb.

Autophagy can act as a defense mechanism for macrophages infected by intracellular pathogens. Mycobacterium tuberculosis (Mtb) is known to both induce and repress autophagic responses, such as xenophagy and LC3-associated phagocytosis (LAP) which both involve the recruitment of LC3 to the Mtb-containing vacuole (MCV). However, the dynamics of MCV interaction with xenophagy or LAP are unclear. Here, using time-lapse confocal microscopy, we present a comprehensive spatio-temporal analysis of the LC3 recruitment to the MCVs during the infection of macrophages. The results revealed frequent LC3 recruitment in the form of large tubule-vesicular structures to the MCV, characteristic of xenophagy, and demonstrated that Mtb could efficiently escape from this signal. We found that the main driver of the LC3 recruitment is the initial macrophage bacterial burden before a second phagocytosis event. We also assessed the potential bactericidal properties of the LC3 recruitment and observed that interferon-gamma treatments did not affect the LC3 recruitment frequency. Additionally, no sign of acidification in the formed autophagosome with or without interferon-gamma treatment was observed. Interestingly, the time-lapses using the acidification probe lysoview revealed that the LC3 recruitment happened shortly after a drop in acidity, a typical sign of membrane damage that is a well-known trigger for autophagy. However, LC3 subsequent loss of signal or escape could also be followed by a restoration of acidification in the vacuole, thus showing restoration of membrane integrity. In conclusion, we show that LC3 recruitment to the MCV correlates with subsequent membrane repair. However, the LC3 recruitment did not show bactericidal properties, questioning its cell intrinsic role in controlling the Mtb infection in macrophages.

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The SWI/SNF complex plays an important role in controlling gene expression via chromatin remodeling. One of its catalytic subunits, SMARCA4, is frequently mutated in multiple tumor types, and SMARCA4-deficient cells are highly dependent on the other catalytic subunit, SMARCA2, for survival. Therefore, targeting SMARCA2 with selective protein degraders has therapeutic potential in SMARCA4 deficient human cancers. We have previously described the development of a potent and selective SMARCA2 targeted degrader, PRT3789, that demonstrates robust degradation of SMARCA2 protein and excellent preclinical efficacy in SMARCA4-del models (Hulse, et al. 2022). PRT3789 will be evaluated in a Phase 1 clinical trial in patients with SMARCA4-mutant cancer. In order to assess target engagement following clinical administration of PRT3789, we developed two pharmacodynamic (PD) assays to measure SMARCA2 protein degradation and changes in SMARCA2 target gene expression in human peripheral mononuclear cells (PBMCs). We used the MSD® S-PLEX platform for the development of a sensitive and quantitative, plate-based immunoassay for determining the protein concentration of SMARCA2 in human PBMC lysates. This assay exhibits a wide dynamic range of detection for SMARCA2 with an LLOD at 1.5 pg/mL, as well as exhibiting acceptable spike recovery and minimal cross-reactivity to SMARCA4. In addition, we optimized the PBMC isolation protocol and lysis conditions to enable sensitive detection of SMARCA2 protein. In order to demonstrate downstream gene expression changes as a consequence of SMARCA2 protein degradation, we developed a secondary qPCR assay. PBMCs from healthy human donors were treated ex vivo with PRT3789 for 24 - 48 hours, followed by RNA-sequencing to identify differentially expressed genes. We refined an 8-gene panel that showed robust expression in PBMCs, as well as consistent and dose-dependent changes in response to PRT3789 treatment. We further calculated a differential gene expression (DGE) score based on expression of this gene panel that correlates with SMARCA2 protein degradation. In summary, we describe the development and characterization of two independent clinically relevant PD assays that can be used to evaluate target engagement in blood samples following PRT3789 administration. Citation Format: Andrew Moore, Carly Bachner, Lalitha Srinivasan, Pradeep Kurup, Alex Grego, Caroline Vitkovitsky, Koichi Ito, Neha Bhagwat, Peggy Scherle. Development of pharmacodynamic assays for quantifying SMARCA2 protein degradation and target gene expression in response to a SMARCA2 degrader (PRT3789) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2792.