Tumor Suppressor Adenomatous Polyposis Coli Sustains Dendritic Cell Tolerance through IL-10 in a ß-Catenin-Dependent Manner.

Dendritic cells (DC) play important roles in balancing immunity and tolerance, in which ß-catenin signaling plays an important role, yet the underlying mechanisms remain elusive. In this study, we investigated the functions of the tumor suppressor adenomatous polyposis coli (APC), also a key component of the ß-catenin upstream destruction complex in DC. APC depletion in DC does not alter DC and T cell homeostasis under resting conditions. However, APC deficiency in DC leads to attenuated antitumor immunity in mice, which exhibit fewer CD8+ T cells and more Foxp3+ regulatory T cells in tumor and draining lymph nodes. Loss of APC in DC does not affect the expression levels of costimulatory molecules. However, APC-deficient DC produce more IL-10 and exhibit a higher ability of inducing regulatory T cells but a lower ability of priming CD8+ T cells, both of which can be reversed by IL-10 inhibition. Lastly, ß-catenin depletion in APC-deficient DC rescues their antitumor immunity and reverses elevated IL-10 production. Taken together, our results identify that APC drives DC tolerance via the ß-catenin/IL-10 axis.

Vps33B controls Treg cell suppressive function through inhibiting lysosomal nutrient sensing complex-mediated mTORC1 activation.

The suppressive function of regulatory T (Treg) cells is tightly controlled by nutrient-fueled mechanistic target of rapamycin complex 1 (mTORC1) activation, yet its dynamics and negative regulation remain unclear. Here we show that Treg-specific depletion of vacuolar protein sorting 33B (Vps33B) in mice results in defective Treg cell suppressive function and acquisition of effector phenotype, which in turn leads to disturbed T cell homeostasis and boosted antitumor immunity. Mechanistically, Vps33B binds with lysosomal nutrient-sensing complex (LYNUS) and promotes late endosome and lysosome fusion and clearance of the LYNUS-containing late endosome/lysosome, and therefore suppresses mTORC1 activation. Vps33B deficiency in Treg cells results in disordered endosome lysosome fusion, which leads to accumulation of LYNUS that causes elevated mTORC1 activation and hyper-glycolytic metabolism. Taken together, our study reveals that Vps33B maintains Treg cell suppressive function through sustaining endolysosomal homeostasis and therefore restricting amino acid-licensed mTORC1 activation and metabolism.

Ubc13 Promotes K63-Linked Polyubiquitination of NLRP3 to Activate Inflammasome.

NLRP3 inflammasome plays an important role in innate immune system through recognizing pathogenic microorganisms and danger-associated molecules. Deubiquitination of NLRP3 has been shown to be essential for its activation, yet the functions of Ubc13, the K63-linked specific ubiquitin-conjugating enzyme E2, in NLRP3 inflammasome activation are not known. In this study, we found that in mouse macrophages, Ubc13 knockdown or knockout dramatically impaired NLRP3 inflammasome activation. Catalytic activity is required for Ubc13 to control NLRP3 activation, and Ubc13 pharmacological inhibitor significantly attenuates NLRP3 inflammasome activation. Mechanistically, Ubc13 associates with NLRP3 and promotes its K63-linked polyubiquitination. Through mass spectrum and biochemical analysis, we identified lysine 565 and lysine 687 as theK63-linked polyubiquitination sites of NLRP3. Collectively, our data suggest that Ubc13 potentiates NLRP3 inflammasome activation via promoting site-specific K63-linked ubiquitination of NLRP3. Our study sheds light on mechanisms of NLRP3 inflammasome activation and identifies that targeting Ubc13 could be an effective therapeutic strategy for treating aberrant NLRP3 inflammasome activation-induced pathogenesis.

SIRT3-mediated deacetylation of NLRC4 promotes inflammasome activation.

Salmonella typhimurium (S. typhimurium) infection of macrophage induces NLRC4 inflammasome-mediated production of the pro-inflammatory cytokines IL-1ß. Post-translational modifications on NLRC4 are critical for its activation. Sirtuin3 (SIRT3) is the most thoroughly studied mitochondrial nicotinamide adenine dinucleotide (NAD+) -dependent deacetylase. We wondered whether SIRT3 mediated-deacetylation could take part in NLRC4 inflammasome activation. Methods: We initially tested IL-1ß production and pyroptosis after cytosolic transfection of flagellin or S. typhimurium infection in wild type and SIRT3-deficient primary peritoneal macrophages via immunoblotting and ELISA assay. These results were confirmed in SIRT3-deficient immortalized bone marrow derived macrophages (iBMDMs) which were generated by CRISPR-Cas9 technology. In addition, in vivo experiments were conducted to confirm the role of SIRT3 in S. typhimurium-induced cytokines production. Then NLRC4 assembly was analyzed by immune-fluorescence assay and ASC oligomerization assay. Immunoblotting, ELISA and flow cytometry were performed to clarify the role of SIRT3 in NLRP3 and AIM2 inflammasomes activation. To further investigate the mechanism of SIRT3 in NLRC4 activation, co-immunoprecipitation (Co-IP), we did immunoblot, cellular fractionation and in-vitro deacetylation assay. Finally, to clarify the acetylation sites of NLRC4, we performed liquid chromatography-mass spectrometry (LC-MS) and immunoblotting analysis. Results: SIRT3 deficiency led to significantly impaired NLRC4 inflammasome activation and pyroptosis both in vitro and in vivo. Furthermore, SIRT3 promotes NLRC4 inflammasome assembly by inducing more ASC speck formation and ASC oligomerization. However, SIRT3 is dispensable for NLRP3 and AIM2 inflammasome activation. Moreover, SIRT3 interacts with and deacetylates NLRC4 to promote its activation. Finally, we proved that deacetylation of NLRC4 at Lys71 or Lys272 could promote its activation. Conclusions: Our study reveals that SIRT3 mediated-deacetylation of NLRC4 is pivotal for NLRC4 activation and the acetylation switch of NLRC4 may aid the clearance of S. typhimurium infection.