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Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

University of Cologne

Riboflavin, also known as vitamin B2 , is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte-specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK-deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF-induced priming of Nox2, which is needed for oxidative burst, was defective in RFK-deficient phagocytes. Lack of riboflavin in wild-type macrophages for only 6 h shut down TNF-induced, RFK-mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti-listerial activity. Vice versa, ROS production by riboflavin-deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo. 

Riboflavin (vitamin B2 ) deficiency impairs NADPH oxidase 2 (Nox2) priming and defense against Listeria monocytogenes.

Use of adeno-associated viral (AAV) vectors for liver-directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)-dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV-mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short-term treatment with a single dose of torin 1 significantly increased vector-mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid-α-glucosidase in nonhuman primates.

Autophagy determines efficiency of liver-directed gene therapy with adeno-associated viral vectors.

The intracellular pathogen Listeria monocytogenes (L.m.) is targeted by the autophagic machinery, but the molecular mechanisms involved and consequences for anti-listerial immunity remain enigmatic. Here, we demonstrate that L.m. infection of macrophages in vivo exclusively evokes LC3-associated phagocytosis (LAP), but not canonical autophagy, and that targeting of L.m. by LAP is required for anti-listerial immunity. The pathway leading to LAP induction in response to L.m. infection emanates from the β integrin Mac-1 (CR3, integrin αβ), a receptor recognizing diverse microbial ligands. Interaction of L.m. with Mac-1 induces acid sphingomyelinase-mediated changes in membrane lipid composition that facilitate assembly and activation of the phagocyte NAPDH oxidase Nox2. Nox2-derived reactive oxygen species then trigger LC3 recruitment to L.m.-containing phagosomes by LAP. By promoting fusion of L.m.-containing phagosomes with lysosomes, LAP increases exposure of L.m. to bactericidal acid hydrolases, thereby enhancing anti-listerial activity of macrophages and immunity of mice.

The β Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes.

The macroautophagic/autophagic machinery cannot only target cell-endogenous components but also intracellular pathogenic bacteria such as Listeria monocytogenes. Listeria are targeted both by canonical autophagy and by a noncanonical form of autophagy referred to as LC3-associated phagocytosis (LAP). The molecular mechanisms involved and whether these processes contribute to anti-listerial immunity or rather provide Listeria with a replicative niche for persistent infection, however, remained unknown. Recently, using an in vivo mouse infection model, we have been able to demonstrate that Listeria in tissue macrophages are targeted exclusively by LAP. Furthermore, our data show that LAP is required for killing of Listeria by macrophages and thereby contributes to anti-listerial immunity of mice, whereas canonical autophagy is completely dispensable. Moreover, we have elucidated the molecular mechanisms that trigger LAP of Listeria and identified the integrin ITGAM-ITGB2/Mac-1/CR3/integrin αß as the receptor that initiates LAP in response to Listeria infection.

LC3-associated phagocytosis initiated by integrin ITGAM-ITGB2/Mac-1 enhances immunity to Listeria monocytogenes.

A major function of macrophages during infection is initiation of the proinflammatory response, leading to the secretion of cytokines that help to orchestrate the immune response. Here, we identify reactive oxygen species (ROS) as crucial mediators of proinflammatory signaling leading to cytokine secretion in infected macrophages. ROS produced by NADPH oxidases (Noxes), such as Nox2, are key components of the macrophage response to invading pathogens; however, our data show that the ROS that mediated proinflammatory signaling were produced by mitochondria (mtROS). We identified the inhibitor of κB (IκB) kinase (IKK) complex regulatory subunit NEMO [nuclear factor κB (NF-κB) essential modulator] as a target for mtROS. Specifically, mtROS induced intermolecular covalent linkage of NEMO through disulfide bonds formed by Cys and Cys, which was essential for activation of the IKK complex and subsequent signaling through the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and NF-κB pathways that eventually led to the secretion of proinflammatory cytokines. We thus identify mtROS-dependent disulfide linkage of NEMO as an essential regulatory step of the proinflammatory response of macrophages to bacterial infection.

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO.

Phagocytes ingest, kill and degrade invading microbes in a process called phagocytosis. LC3-associated phagocytosis (LAP) combines the molecular machinery of phagocytosis with that of autophagy, the cellular pathway for ingestion of cytoplasmic components, resulting in the eponymous association of 'microtubule-associated proteins 1 A/1B light chain 3' (LC3) with the phagosomal membrane. The LC3-decorated phagosomes, or LAPosomes, show enhanced fusion with lysosomes resulting in enhanced killing and degradation of contained pathogens. Thus, LAP is a particularly microbicidal pathway. In this review, we discuss the molecular mechanisms involved in induction and execution of LAP and its crucial role in antimicrobial immunity against bacteria, fungi and parasites. As LAP has only recently been defined, we also point out the key open questions that remain to be answered.

Alexander Gluschko

Institute for Medical Microbiology,
Immunology and Hygiene,
Faculty of Medicine and University Hospital Cologne,
Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne

University of Cologne

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA