Showing papers by "Alan Sher published in 2018"

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.

Host resistance to pulmonary Mycobacterium tuberculosis infection requires CD153 expression.

Abstract: Mycobacterium tuberculosis infection (Mtb) is the leading cause of death due to a single infectious agent and is among the top ten causes of all human deaths worldwide1 CD4 T cells are essential for resistance to Mtb infection, and for decades it has been thought that IFNγ production is the primary mechanism of CD4 T-cell-mediated protection2,3 However, IFNγ responses do not correlate with host protection, and several reports demonstrate that additional anti-tuberculosis CD4 T-cell effector functions remain unaccounted for4-8 Here we show that the tumour-necrosis factor (TNF) superfamily molecule CD153 (encoded by the gene Tnfsf8) is required for control of pulmonary Mtb infection by CD4 T cells In Mtb-infected mice, CD153 expression is highest on Mtb-specific T helper 1 (TH1) cells in the lung tissue parenchyma, but its induction does not require TH1 cell polarization CD153-deficient mice develop high pulmonary bacterial loads and succumb early to Mtb infection Reconstitution of T-cell-deficient hosts with either Tnfsf8-/- or Ifng-/- CD4 T cells alone fails to rescue mice from early mortality, but reconstitution with a mixture of Tnfsf8-/- and Ifng-/- CD4 T cells provides similar protection as wild-type T cells In Mtb-infected non-human primates, CD153 expression is much higher on Ag-specific CD4 T cells in the airways compared to blood, and the frequency of Mtb-specific CD153-expressing CD4 T cells inversely correlates with bacterial loads in granulomas In Mtb-infected humans, CD153 defines a subset of highly polyfunctional Mtb-specific CD4 T cells that are much more abundant in individuals with controlled latent Mtb infection compared to those with active tuberculosis In all three species, Mtb-specific CD8 T cells did not upregulate CD153 following peptide stimulation Thus, CD153 is a major immune mediator of host protection against pulmonary Mtb infection and CD4 T cells are one important source of this molecule

Foreign antigen-independent memory-phenotype CD4 + T cells: a new player in innate immunity?

Abstract: Foreign antigen-independent memory-phenotype CD4 + T cells: a new player in innate immunity?

Inhibition of mTORC1 activity is required for the IL-12p40 response of classical CD16neg CD14+ primary human monocytes to Toxoplasma gondii

Abstract: Primary human monocytes produce IL-12 and TNF in response to live Toxoplasma gondii tachyzoytes by a mechanism requiring parasite phagocytosis. Interestingly, in contrast to the patrolling CD16 + CD14 + subset, monocytes belonging to the major CD16 neg CD14 + subset fail to secrete IL-12 and TNF following T. gondii exposure. Here we show that the CD16 neg CD14 + subset, nevertheless, upregulates Il12b mRNA and thus is able to sense the parasite. In addition, we demonstrate that priming with IFNg, but not IFNa, enables CD16 neg CD14 + monocytes to produce IL-12 protein in response to T. gondii . This effect of IFNg priming was not due to augmented phagocytosis and was not associated with altered rates of glycolysis, oxygen consumption, or increased Il12b mRNA expression. To test the possible role of IFNγ on cytokine translation, we evaluated the requirement for mTOR (mTORC1/2) pathway, a key regulator of protein synthesis. The T. gondii -induced IL-12 response of IFNγ primed CD16 neg monocytes was inhibited by Torin, an inhibitor of mTORC2, but surprisingly not by the classical mTORC1 inhibitor Rapamycin. Because mTORC2 has been shown to downregulate mTORC1 activity, we hypothesized that the priming effect of IFNg might result from the upregulation of mTORC2 resulting in suppressed mTORC1. Indeed, in the absence of IFNγ, suppression of mTORC1 in CD16 neg monocytes with Rapamycin enhanced IL-12 secretion. Taken together, our results suggest that classical CD16 neg CD14 + monocytes fail to secrete IL-12 in response to T. gondii due to increased mTORC1 activity, which can be reversed directly by Rapamycin or indirectly through stimulation of mTORC2 by IFNγ priming.