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.

Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection

“…The ability to disrupt phagosomes seems important for inflammasome activation by Mtb, and it has been reported that phagosomal acidification is a prerequisite for the membrane damaging activity of ESX-1 63–65 . Additionally, active cathepsin release from ruptured phagolysosomes is a suggested trigger for the NLRP3 inflammasome 24, 39, 40 . We therefore went on to investigate the possible involvement of phagosomal acidification and release of active cathepsins in inflammasome activation and pyroptosis by Mtb.…”

“…Lysosomal damaging agents are one of the most clinically relevant classes of NLRP3 triggers 83 . Cathepsin release from damaged lysosomes, especially of Cathepsin B, is one commonly proposed connection, although how this further causes K+ efflux has been unclear 24, 39, 40 . One main source of this confusion is the widespread use of the Cathepsin B inhibitor Ca-074-Me, which appears to inhibit NLRP3 activation independent of Cathepsin B inhibition (our results and 28, 41–43) .…”

“…ESX-1 secretes a range of protein substrates, and is postulated to mediate phagosome permeabilization with full or partial translocation of Mtb into the cytosol 35–38 . Phagosomal permeabilization presumably allows the release of Mtb DNA and activation of AIM2 19 , and release of active cathepsin B from damaged phagolysosomes has been proposed as a trigger for NLRP3 24, 39, 40 . However, off target inhibitor effects and negative results in knockout experiments have left the jury open on the role of cathepsin release in NLRP3 inflammasome activation 28, 41–43 .…”

Mycobacterium tuberculosisstimulates IL-1β production by macrophages in an ESAT-6 dependent manner with the involvement of serum amyloid A3

“…A variety of human pathogens, including Mtb (36) stimulate the production of IL-1β by macrophages and other immune cells, such as dendritic cells and neutrophils. Previous studies have shown that Mtb stimulates IL-1β production by macrophages with the involvement of activated NLRP3, caspase 1 and cathepsin B in an ESAT-6 secretion system (ESX)-1 dependent manner (17, 18). In this study, we have demonstrated with data from both in vitro macrophage infection with Mtb and in vivo mouse intranasal treatment with ESAT-6 or infection with Mtb that ESAT-6 is required for Mtb- induced mature IL-1β production by macrophages with the involvement of K+ efflux and activated NLRP3 (17, 22).…”

Caracterização genética e funcional do inflamassoma na resposta à micobactéria e no desenvolvimento de diferentes formas clínicas de tuberculose pulmonar

“…Levando em conta que (AMARAL, E. P. et al, 2018) demonstraram, em modelo murino de TB que a liberação de catepsinas lisossomais é um evento chave na ativação de NLRP3 por Mtb, testamos essa via no nosso modelo de MDM humanos e confirmamos que a liberação de catepsina B é um evento chave na ativação do inflamassoma frente a Mtb, e que esse mecanismo possivelmente não afeta apenas a ativação do NLRP3 mas também de outros receptores, sendo que o efeito inibitório é maior quando comparado a inibição especifica do NLRP3. É interessante destacar que foi reportado recentemente que a liberação de catepsinas ativa o NLRC4 em um modelo murino de infecção por Salmonella (LAGE et al, 2013), sugerindo que, como ja observado por (MASTER et al, 2008) do epitélio e na polarização dos linfócitos T CD4 + em células efetoras Th1 produtoras de IFN-γ.…”