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Following detection of pathogen infection and disrupted cellular homeostasis, cells can activate a range of cell death pathways, such as apoptosis, necroptosis and pyroptosis, as part of their defence strategy. The initiation of pro-inflammatory, lytic pyroptosis is controlled by inflammasomes, which respond to a range of cellular perturbations. As is true for many host defence pathways, pathogens have evolved multiple mechanisms to subvert this pathway, many of which have only recently been described. Herein, we will discuss the mechanisms by which inflammasomes sense pathogen invasion and initiate pyroptosis and the effector mechanisms used by pathogens to suppress this pathway and preserve their niche.

Autophagy is a cellular innate-immune defence mechanism against intracellular microorganisms, including Mycobacterium tuberculosis (Mtb). How canonical and non-canonical autophagy function to control Mtb infection in phagosomes and the cytosol remains unresolved. Macrophages are the main host cell in humans for Mtb. Here we studied the contributions of canonical and non-canonical autophagy in the genetically tractable human induced pluripotent stem cell-derived macrophages (iPSDM), using a set of Mtb mutants generated in the same genetic background of the common lab strain H37Rv. We monitored replication of Mtb mutants that are either unable to trigger canonical autophagy (Mtb ΔesxBA) or reportedly unable to block non-canonical autophagy (Mtb ΔcpsA) in iPSDM lacking either ATG7 or ATG14 using single-cell high-content imaging. We report that deletion of ATG7 by CRISPR–Cas9 in iPSDM resulted in increased replication of wild-type Mtb but not of Mtb ΔesxBA or Mtb ΔcpsA. We show that deletion of ATG14 resulted in increased replication of both Mtb wild type and the mutant Mtb ΔesxBA. Using Mtb reporters and quantitative imaging, we identified a role for ATG14 in regulating fusion of phagosomes containing Mtb with lysosomes, thereby enabling intracellular bacteria restriction. We conclude that ATG7 and ATG14 are both required for restricting Mtb replication in human macrophages.

SummaryThe intracellular population of Mycobacterium tuberculosis (Mtb) is dynamically segregated within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a dual-imaging approach that allows quantitative monitoring of host subcellular acidification and Mtb intrabacterial pH profiles by live-fluorescence microscopy in a biosafety level 3 laboratory. By combining this live imaging approach with pharmacological and genetic perturbations, we show that Mtb can maintain its intracellular pH independently of the surrounding pH in primary human macrophages. Importantly, we show that unlike bedaquiline (BDQ), isoniazid (INH) or rifampicin (RIF), the front-line drug pyrazinamide (PZA) displays antibacterial efficacy by acting as protonophore which disrupts intrabacterial pH homeostasis in cellulo. By using Mtb mutants with different intra-macrophage localisation, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this dual imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens.HighlightsMtb maintains its intrabacterial pH inside both acidic and neutral subcellular microenvironments of human macrophagesPyrazinamide, but not other frontline antibiotics, acts as a protonophore in celluloPyrazinamide-mediated intrabacterial pH homeostasis disruption and antibacterial efficacy requires host endolysosomal acidificationCytosolic localisation mediated by ESX-1 contributes to pyrazinamide antibacterial activity resistancePyrazinamide conversion into pyrazinoic acid by the pyrazinamidase/nicotinamidase PncA is essential for its protonophore activity and efficacy in cellulo

Cells respond to endolysosome damage by either repairing the damage or targeting damaged endolysosomes for degradation via lysophagy. However, the signals regulating the decision for repair or lysophagy are poorly characterised. Here, we show that the Parkinson's disease (PD)‐related kinase LRRK2 is activated in macrophages by pathogen‐ or sterile‐induced endomembrane damage. LRRK2 recruits the Rab GTPase Rab8A to damaged endolysosomes as well as the ESCRT‐III component CHMP4B, thereby favouring ESCRT‐mediated repair. Conversely, in the absence of LRRK2 and Rab8A, damaged endolysosomes are targeted to lysophagy. These observations are recapitulated in macrophages from PD patients where pathogenic LRRK2 gain‐of‐function mutations result in the accumulation of endolysosomes which are positive for the membrane damage marker Galectin‐3. Altogether, this work indicates that LRRK2 regulates endolysosomal homeostasis by controlling the balance between membrane repair and organelle replacement, uncovering an unexpected function for LRRK2, and providing a new link between membrane damage and PD., LRRK2 phosphorylation of Rab8A GTPase promotes its co‐translocation with ESCRT component CHMP4B to damaged endolysosomes for their repair.

Antibiotics are widely used in the treatment of bacterial infections. Although known for their microbicidal activity, antibiotics may also interfere with the host’s immune system. Here we analyzed the effects of bedaquiline (BDQ), an inhibitor of the mycobacterial ATP synthase, on human macrophages. Genome-wide gene expression analysis revealed that BDQ reprogramed macrophages into potent bactericidal phagocytes. We found that 1,495 genes were differentially expressed in M. tuberculosis-infected macrophages incubated with the drug, with an over-representation of genes involved in metabolism, lysosome biogenesis and activation. BDQ treatment triggered a variety of antimicrobial defense mechanisms, including nitric oxide production, phagosome-lysosome fusion, and autophagy. These effects were associated with activation of transcription factor EB (TFEB), involved in the transcription of lysosomal genes, resulting in enhanced intracellular killing of different bacterial species that were naturally insensitive to BDQ. Thus, BDQ could be used as a host-directed therapy against a wide range of bacterial infections.

The success of tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb), relies on the ability to survive in host cells and escape to immune surveillance and activation. We recently demonstrated that Mtb manipulation of host lysosomal cathepsins in macrophages leads to decreased enzymatic activity and pathogen survival. In addition, while searching for microRNAs (miRNAs) involved in posttranscriptional gene regulation during mycobacteria infection of human macrophages, we found that selected miRNAs such as miR-106b-5p were specifically upregulated by pathogenic mycobacteria. Here, we show that miR-106b-5p is actively manipulated by Mtb to ensure its survival in macrophages. Using an in silico prediction approach, we identified miR-106b-5p with a potential binding to the 3'-untranslated region of cathepsin S (CtsS) mRNA. We demonstrated by luminescence-based methods that miR-106b-5p indeed targets CTSS mRNA resulting in protein translation silencing. Moreover, miR-106b-5p gain-of-function experiments lead to a decreased CtsS expression favoring Mtb intracellular survival. By contrast, miR-106b-5p loss-of-function in infected cells was concomitant with increased CtsS expression, with significant intracellular killing of Mtb and T-cell activation. Modulation of miR-106b-5p did not impact necrosis, apoptosis or autophagy arguing that miR-106b-5p directly targeted CtsS expression as a way for Mtb to avoid exposure to degradative enzymes in the endocytic pathway. Altogether, our data suggest that manipulation of miR-106b-5p as a potential target for host-directed therapy for Mtb infection.

Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defenses. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation.

Xenophagy is an important cellular defence mechanism against cytosol invading pathogens, such as Mycobacterium tuberculosis (Mtb). Activation of xenophagy in macrophages targets Mtb to autophagosomes, however how Mtb is targeted to autophagosomes in human macrophages at a high spatial and temporal resolution is unknown. Here, we use human induced pluripotent stem cell derived macrophages (iPSDM) to study the human macrophage response to Mtb infection induced by the ESX-1 Type-VII secretion system. Using RNA-seq, we identify ESX-1 dependent transcriptional responses in iPSDM after infection with Mtb. This analysis revealed differential inflammatory responses and dysregulated pathways such as Eukaryotic Initiation Factor 2 (eIF2) signalling and protein ubiquitination. Moreover, live cell imaging revealed that Mtb infection in human macrophages induces dynamic ESX-1-dependent, LC3B positive tubulovesicular autophagosomes (LC3-TVS). Through a correlative live cell/FIB SEM approach, we show that upon phagosomal rupture Mtb induces the formation of LC3-TVS, from which it is able to escape to reside in the cytosol. Thus, iPSDM represent a valuable model for studying spatiotemporal dynamics of human macrophage-Mtb interactions and that Mtb is able to evade capture by autophagic compartments.

Mitochondrial dynamics and metabolism are closely associated, however, if these interactions play a role in the human macrophage response to Mycobacterium tuberculosis (Mtb) remain largely unknown. Here, human induced-pluripotent stem cell derived macrophages (iPSDM) were used to define whether mitochondrial dynamics and metabolism are modulated by Mtb infection. Macrophage bioenergetic response to infection was analysed by extracellular flux analysis (Seahorse) and unbiased metabolomics. In a complementary approach, high-content live cell imaging and single cell analysis was used to investigate changes in mitochondrial dynamics and Mtb intracellular replication. The metabolic profile of infected human macrophages showed an increase in the oxygen consumption and extracellular acidification rate after 48h of infection. Moreover, the lipidomics analysis showed a significant upregulation in the total levels of sphingomyelin and ceramide. Interestingly, these changes in metabolism were not associated with a disruption of the mitochondrial network. However, infection decreased mitochondrial protein levels, which correlated with RNA-Seq analysis showing downregulation of mitochondrial transcripts. The inhibition of glycolysis with 2-Deoxy-d-glucose promoted Mtb replication, whereas oxamate (a lactate dehydrogenase inhibitor) impaired Mtb growth. Our results show that macrophage metabolic reprogramming is required for the control of Mtb replication. Unlike other intracellular pathogens, Mtb did not induce fragmentation of the mitochondrial network, suggesting that manipulation of host cell metabolism might correlate with disruption of mitochondrial function rather than morphology.

Mitochondrial dynamics and metabolism in immune cells are closely associated. However, if these interactions play a role in the human macrophage response to Mycobacterium tuberculosis (Mtb) remains largely unknown. We used human induced-pluripotent stem cell derived macrophages (iPSDM) together with high-content live-cell imaging, extracellular flux analysis and unbiased metabolomics to investigate mitochondrial dynamics, Mtb intracellular replication and mitochondrial metabolism. Mitochondrial tracking at the single-cell level revealed extensive mitochondrial morphology heterogeneity. Surprisingly, Mtb did not induce significant changes in the mitochondrial area, length or width during the first 48h of infection. We did not find a correlation between Mtb intracellular replication and changes in mitochondrial morphological parameters. However, Mtb induced a decrease in the mitochondrial membrane potential and protein turnover rate, as visualised by MitoTracker Red and the ratiometric reporter MitoTimer, respectively. The metabolic profile of infected macrophages showed an increase in oxygen consumption and extracellular acidification rate after 48h of infection. These changes were associated with decreased mitochondrial protein and transcripts levels. The inhibition of glycolysis with 2-Deoxy-d-glucose promoted Mtb replication, whereas oxamate (lactate dehydrogenase inhibitor) impaired Mtb intracellular replication. Collectively, our results show that macrophage metabolic reprogramming is required for the control of Mtb replication. Unlike other intracellular pathogens, changes in host cell metabolism induced by Mtb might correlate with disruption of mitochondrial function rather than morphology.