HDAC7 is an immunometabolic switch triaging danger signals for engagement of antimicrobial versus inflammatory responses in macrophages.

The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1ß production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.

TRIM21 Is Targeted for Chaperone-Mediated Autophagy during Salmonella Typhimurium Infection.

Salmonella enterica serovar Typhimurium (S Typhimurium) is a Gram-negative bacterium that induces cell death of macrophages as a key virulence strategy. We have previously demonstrated that the induction of macrophage death is dependent on the host's type I IFN (IFN-I) response. IFN-I signaling has been shown to induce tripartite motif (TRIM) 21, an E3 ubiquitin ligase with critical functions in autoimmune disease and antiviral immunity. However, the importance and regulation of TRIM21 during bacterial infection remains poorly understood. In this study, we investigated the role of TRIM21 upon S Typhimurium infection of murine bone marrow-derived macrophages. Although Trim21 expression was induced in an IFN-I-dependent manner, we found that TRIM21 levels were mainly regulated posttranscriptionally. Following TLR4 activation, TRIM21 was transiently degraded via the lysosomal pathway by chaperone-mediated autophagy (CMA). However, S Typhimurium-induced mTORC2 signaling led to phosphorylation of Akt at S473, which subsequently impaired TRIM21 degradation by attenuating CMA. Elevated TRIM21 levels promoted macrophage death associated with reduced transcription of NF erythroid 2-related factor 2 (NRF2)-dependent antioxidative genes. Collectively, our results identify IFN-I-inducible TRIM21 as a negative regulator of innate immune responses to S Typhimurium and a previously unrecognized substrate of CMA. To our knowledge, this is the first study reporting that a member of the TRIM family is degraded by the lysosomal pathway.

Variation in hemolysin A expression between uropathogenic Escherichia coli isolates determines NLRP3-dependent vs. -independent macrophage cell death and host colonization.

Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections (UTIs). The multidrug-resistant E. coli sequence type 131 (ST131) clone is a serious threat to human health, yet its effects on immune responses are not well understood. Here we screened a panel of ST131 isolates, finding that only strains expressing the toxin hemolysin A (HlyA) killed primary human macrophages and triggered maturation of the inflammasome-dependent cytokine IL-1ß. Using a representative strain, the requirement for the hlyA gene in these responses was confirmed. We also observed considerable heterogeneity in levels of cell death initiated by different HlyA+ve ST131 isolates, and this correlated with secreted HlyA levels. Investigation into the biological significance of this variation revealed that an ST131 strain producing low levels of HlyA initiated cell death that was partly dependent on the nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, with this response being associated with a host-protective role in a mouse UTI model. When the same ST131 strain was engineered to overexpress high HlyA levels, macrophage cell death occurred even when NLRP3 function was abrogated, and bladder colonization was significantly increased. Thus, variation in HlyA expression in UPEC affects mechanisms by which macrophages die, as well as host susceptibility vs. resistance to colonization.-Murthy, A. M. V., Sullivan, M. J., Nhu, N. T. K., Lo, A. W., Phan, M.-D., Peters, K. M., Boucher, D., Schroder, K., Beatson, S. A., Ulett, G. C., Schembri, M. A., Sweet, M. J. Variation in hemolysin A expression between uropathogenic Escherichia coli isolates determines NLRP3-dependent vs. -independent macrophage cell death and host colonization.

Crosstalk between cGAS-STING signaling and cell death.

Cytosolic nucleic acid sensors have a critical role in detecting endogenous nucleic acids to initiate innate immune responses during microbial infections and/or cell death. Several seminal studies over the past decade have delineated the conserved mechanism of cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) and the downstream signaling adapter stimulator of interferon genes (STING) in mediating innate immune signaling pathways as a host defense mechanism. Besides the predominant role in microbial infections and inflammatory diseases, there is an increased attention on alternative functional responses of cGAS-STING-mediated signaling. Here we review the complexity of interactions between the cGAS-STING signaling and cell death pathways. A better understanding of molecular mechanisms of this interplay is important with regard to the development of new therapeutics targeting cGAS-STING signaling in cancer, infectious, and chronic inflammatory diseases.

Class IIa Histone Deacetylases Drive Toll-like Receptor-Inducible Glycolysis and Macrophage Inflammatory Responses via Pyruvate Kinase M2.

Histone deacetylases (HDACs) drive innate immune cell-mediated inflammation. Here we identify class IIa HDACs as key molecular links between Toll-like receptor (TLR)-inducible aerobic glycolysis and macrophage inflammatory responses. A proteomic screen identified the glycolytic enzyme pyruvate kinase M isoform 2 (Pkm2) as a partner of proinflammatory Hdac7 in murine macrophages. Myeloid-specific Hdac7 overexpression in transgenic mice amplifies lipopolysaccharide (LPS)-inducible lactate and promotes a glycolysis-associated inflammatory signature. Conversely, pharmacological or genetic targeting of Hdac7 and other class IIa HDACs attenuates LPS-inducible glycolysis and accompanying inflammatory responses in macrophages. We show that an Hdac7-Pkm2 complex acts as an immunometabolism signaling hub, whereby Pkm2 deacetylation at lysine 433 licenses its proinflammatory functions. Disrupting this complex suppresses inflammatory responses in vitro and in vivo. Class IIa HDACs are thus pivotal intermediates connecting TLR-inducible glycolysis to inflammation via Pkm2.

Complex Multilevel Control of Hemolysin Production by Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections. Nearly half of all UPEC strains secrete hemolysin, a cytotoxic pore-forming toxin. Here, we show that the prevalence of the hemolysin toxin gene (hlyA) is highly variable among the most common 83 E. coli sequence types (STs) represented on the EnteroBase genome database. To explore this diversity in the context of a defined monophyletic lineage, we contextualized sequence variation of the hlyCABD operon within the genealogy of the globally disseminated multidrug-resistant ST131 clone. We show that sequence changes in hlyCABD and its newly defined 1.616-kb-long leader sequence correspond to phylogenetic designation, and that ST131 strains with the strongest hemolytic activity belong to the most extensive multidrug-resistant sublineage (clade C2). To define the set of genes involved in hemolysin production, the clade C2 strain S65EC was completely sequenced and subjected to a genome-wide screen by combining saturated transposon mutagenesis and transposon-directed insertion site sequencing with the capacity to lyse red blood cells. Using this approach, and subsequent targeted mutagenesis and complementation, 13 genes were confirmed to be specifically required for production of active hemolysin. New hemolysin-controlling elements included discrete sets of genes involved in lipopolysaccharide (LPS) inner core biosynthesis (waaC, waaF, waaG, and rfaE) and cytoplasmic chaperone activity (dnaK and dnaJ), and we show these are required for hemolysin secretion. Overall, this work provides a unique description of hemolysin sequence diversity in a single clonal lineage and describes a complex multilevel system of regulatory control for this important toxin.IMPORTANCE Uropathogenic E. coli (UPEC) is the major cause of urinary tract infections and a frequent cause of sepsis. Nearly half of all UPEC strains produce the potent cytotoxin hemolysin, and its expression is associated with enhanced virulence. In this study, we explored hemolysin variation within the globally dominant UPEC ST131 clone, finding that strains from the ST131 sublineage with the greatest multidrug resistance also possess the strongest hemolytic activity. We also employed an innovative forward genetic screen to define the set of genes required for hemolysin production. Using this approach, and subsequent targeted mutagenesis and complementation, we identified new hemolysin-controlling elements involved in LPS inner core biosynthesis and cytoplasmic chaperone activity, and we show that mechanistically they are required for hemolysin secretion. These original discoveries substantially enhance our understanding of hemolysin regulation, secretion and function.