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.

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.

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.

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.