Lysosomal Cathepsin Release Is Required for NLRP3-Inflammasome Activation by Mycobacterium tuberculosis in Infected Macrophages

Abstract

Lysosomal cathepsin B (CTSB) has been proposed to play a role in the induction of acute inflammation. We hypothesised that the presence of active CTSB in the cytosol is crucial for NLRP3-inflammasome assembly and, consequently, for mature IL-1β generation after mycobacterial infection in vitro. Elevated levels of CTSB was observed in the lungs of mice and rabbits following infection with Mycobacterium tuberculosis (Mtb) H37Rv as well as in plasma from acute tuberculosis patients. H37Rv-infected murine bone marrow-derived macrophages (BMDMs) displayed both lysosomal leakage, with release of CTSB into the cytosol, as well as increased levels of mature IL-1β. These responses were diminished in BMDM infected with a mutant H37Rv deficient in ESAT-6 expression. Pharmacological inhibition of cathepsin activity with CA074-Me resulted in a substantial reduction of both mature IL-1β production and caspase-1 activation in infected macrophages. Moreover, cathepsin inhibition abolished the interaction between NLRP3 and ASC, measured by immunofluorescence imaging in H37Rv-infected macrophages, demonstrating a critical role of the enzyme in NLRP3-inflammasome activation. These observations suggest that during Mtb infection, lysosomal release of activated CTSB and possibly other cathepsins inhibitable by CA07-Me is critical for the induction of inflammasome-mediated IL-1β processing by regulating NLRP3-inflammasome assembly in the cytosol.

Keywords: ESAT-6 secretion system; IL-1β; cathepsin B; inflammasome; tuberculosis.

Figure 1

Mycobacterium tuberculosis (Mtb) infection upregulates both cathepsin B (CTSB) expression and enzymatic activity. (A) CTSB activity and (B) expression of mature CTSB were measured in the lungs of H37Rv-infected mice after 1 month or 6 months post-infection (mean ± SEM, n = 5). (C) CTSB activity (red) in macrophages infected (multiplicity of infection: 3) with GFP-H37Rv Mtb strain (green) was analyzed by confocal microscopy (magnification: 600×). DAPI (blue) was used for nuclear staining. Representative images are shown. (D) CTSB messenger RNA expression was quantified in lung granulomatous lesions from rabbits infected with Mtb H37Rv at day 42 post-infection (mean ± SEM, n = 3–4). (E) Total CTSB levels in plasma from patients with active pulmonary TB (PTB; mean ± SEM, n = 13) and health controls (HC; mean ± SEM, n = 12). Data were analyzed using the Mann–Whitney U test (*p < 0.05; **p < 0.01). Data are representative of at least two separate experiments performed.

Figure 2

Mycobacterium tuberculosis (Mtb) infection induces lysosomal leakage dependent on ESAT6 and leads to release of mature cathepsin B (CTSB) and IL-1β production. Cells were stained with lysotracker and mitotracker to verify lysosomal leakage or outer mitochondrial membrane potential disturbance at 24 h p.i. with Mtb H37Rv strain (MOI of 3), respectively. (A) Cells were stimulated with Leu-o-Leu as a positive control of lysosomal leakage. (B) Bone marrow-derived macrophages (BMDMs) were infected with H37Rv, ΔESAT6 H37Rv, or ΔRD1 H37Rv Mtb strains at MOI of 3 as described in Section “Materials and Methods.” Mtb H37Rv induced lysosomal leakage dependent of ESAT6. (C) Cytosolic fraction from BMDMs infected with Mtb H37Rv, showing the presence of mature CTSB. (D,E) BMDMs were infected with H37Rv, ΔESAT6 H37Rv, or ΔRD1 H37Rv strains at MOI of 1 or MOI of 3 as described in Section “Materials and Methods.” (D) IL-1β production was measured by ELISA and (E) expression of mature IL-1β, activated caspases-1, and mature CTSB in the supernatant of cells was assessed by western blotting. Differences were analyzed using the Mann–Whitney U test (between two groups) or the Kruskal–Wallis test with Dunn’s multiple comparisons ad hoc test. Significant differences were observed for the indicated experimental conditions (*p < 0.05; **p < 0.01). Bars and line represent mean and SEM, respectively. Data are representative of three independent experiments using triplicate biological samples.

Figure 3

Mycobacterium tuberculosis (Mtb) ESAT-6-induced IL-1β production is diminished by cathepsin B inhibition. Macrophages were infected with Mtb H37Rv or ΔESAT6 strains at MOI of 3 as described in Section “Materials and Methods.” (A,B) IL-1β production was measured in the supernatants collected from Mtb-infected deficient macrophages or following different treatments as indicated. (B) Mature IL-1β and cleaved caspase-1 were analyzed by western blotting. (C,D) Infected macrophages were stimulated with PA (20 µg/mL) or LFn-ESAT6/PA (20 µg/mL) and IL-1β production was quantified by ELISA after 24 h of infection. (E,F) Bone marrow-derived macrophages were primed with LPS (0.2 µg/mL) for 1 h and further stimulated or not with LFn-ESAT/PA (10 µg/mL each) for 3 h. Furthermore, cells were stimulated with eATP (1 mM) for 25 min and IL-1β production was measured in culture supernatants. Statistical differences observed are shown for each indicated groups (***p < 0.001). The data represent the mean ± SEM of samples in triplicate. Data are from at least three independent experiments.

Figure 4

Cathepsin B (CTSB) activity mediates ASC speck formation. Macrophages were infected with Mycobacterium tuberculosis H37Rv MOI of 3 as described in Section “Materials and Methods” and cells were treated or not with the CTSB inhibitor CA074-Me as indicated. (A) ASC speck formation in infected macrophages treated or not with CA074-Me (25 µM) was analyzed by fluorescence microscopy. (B) The frequency of cells ASC speck positives (C) and number of ASC speck were quantified by using the Image J software. Differences were analyzed using the Mann–Whitney U test. Statistical differences observed are shown for each indicated groups (***p < 0.001). Data represent mean ± SEM of samples ran in triplicate. Results are representative at least two independent experiments.

Figure 5

Schematic model for mature IL-1β generation during Mycobacterium tuberculosis (Mtb) infection. The secretion of ESAT-6 in phagosomes by Mtb induces pore formation on phagosomal membranes, facilitating the leakage of ESAT-6 into cytosol and bacterial escape to cytosolic compartment (A). Once in the cytosol, ESAT-6 may induce pore formation on cellular membranes which could contribute to lysosomal perturbation (LMP) (B). Simultaneously, extracellular ATP is recognized by P2X7 receptor and amplify LMP-induced by Mtb or vice versa (C). LMP releases lysosomal enzymes, such as cathepsin B (CTSB) (D). Once in the cytosol, activated CTSB supports the interaction between NLRP3 and ASC (E) facilitating the NLRP3-inflammasome assembly following caspase-1 recruitment (F). Several molecules of ASC and caspase-1 will be recruited to NLRP3-complex leading the formation of the ASC speck core (G), which amplifies IL-1 β production (H). The secretion of ASC specks and IL-1 β to extracellular milieu may exacerbate inflammation (I).