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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 cells into potent bactericidal phagocytes. We found that 579 and 1,495 genes were respectively differentially expressed in naive- and M. tuberculosis-infected macrophages incubated with the drug, with an over-representation of lysosome-associated genes. BDQ treatment triggered a variety of antimicrobial defense mechanisms, including phagosome-lysosome fusion, and autophagy. These effects were associated with activation of transcription factor EB, 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., eLife digest The discovery of antibiotic drugs, which treat diseases caused by bacteria, has been a hugely valuable advance in modern medicine. They work by targeting specific cellular processes in bacteria, ultimately stopping them from multiplying or killing them outright. Antibiotics sometimes also affect their human hosts and can cause side-effects, such as gut problems or skin reactions. Recent evidence suggests that antibiotics also have an impact on the human immune system. This may happen either indirectly, by affecting ‘friendly’ bacteria normally present in the body, or through direct effects on immune cells. In turn, this could change the effectiveness of drug treatments. For example, if an antibiotic weakens immune cells, the body could have difficulty fighting off the existing infection – or become more vulnerable to new ones. However, even though new drugs are being introduced to combat the worldwide rise of antibiotic-resistant bacteria, their effects on immunity are still not well understood. For example, bedaquiline is an antibiotic recently developed to treat tuberculosis infections that are resistant to several drugs. Giraud-Gatineau et al. wanted to determine if bedaquiline altered the human immune response to bacterial infection independently from its direct anti-microbial effects. Macrophages engulf foreign particles like bacteria and break them down using enzymes stored within small internal compartments, or ‘lysosomes’. Initial experiments using human macrophages, grown both with and without bedaquiline, showed that the drug did not harm the cells and that they grew normally. A combination of microscope imaging and genetic analysis revealed that exposure to bedaquiline not only increased the number of lysosomes within macrophage cells, but also the activity of genes and proteins that increase lysosomes’ ability to break down foreign particles. These results suggested that bedaquiline treatment might make macrophages better at fighting infection, even if the drug itself had no direct effect on bacterial cells. Further studies, where macrophages were first treated with bedaquiline and then exposed to different types of bacteria known to be resistant to the drug, confirmed this hypothesis: in every case, the treated macrophages became efficient bacterial killers. In contrast, older anti-tuberculosis drugs did not have any such potentiating effect on the macrophages. This work sheds new light on our how antibiotic drugs can interact with the cells of the human immune system, and can sometimes even boost our innate defences. Such immune-boosting effects could one day be exploited to make more effective treatments against 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.

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 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.