Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway.

BACKGROUND: NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat. METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study. RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst. CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.

LC3-associated phagocytosis - The highway to hell for phagocytosed microbes.

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.

Co-inhibition of PGF and VEGF blocks their expression in mononuclear phagocytes and limits neovascularization and leakage in the murine retina.

BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of visual impairment in the elderly. The neovascular (wet) form of AMD can be treated with intravitreal injections of different anti-vascular endothelial growth factor (VEGF) agents. Placental growth factor (PGF) is another member of the VEGF family of cytokines with pro-angiogenic and pro-inflammatory effects. Here, we aimed to compare single and combined inhibition of VEGF-A and PGF in the laser-induced mouse model of choroidal neovascularization (CNV) with a focus on the effects on retinal mononuclear phagocytes. METHODS: CNV was induced in C57BL/6J mice using a YAG-Laser. Immediately after laser damage antibodies against VEGF-A (aVEGF), anti-PGF (aPGF), aVEGF combined with aPGF, aflibercept, or IgG control were injected intravitreally in both eyes. Three and 7 days after laser damage, the vascular leakage was determined by fluorescence angiography. Lectin staining of retinal and RPE/choroidal flat mounts was used to monitor CNV. In situ mRNA co-expression of Iba1, VEGF and PGF were quantified using in situ hybridization. Retinal and RPE/choroidal protein levels of VEGF and PGF as well as the pro-inflammatory cytokines IL-6, IL1-beta, and TNF were determined by ELISA. RESULTS: Early (day 3) and intermediate (day 7) vascular leakage and CNV were significantly inhibited by PGF and VEGF-A co-inhibition, most effectively with the trap molecule aflibercept. While VEGF-A blockage alone had no effects, trapping PGF especially with aflibercept prevented the accumulation of reactive microglia and macrophages in laser lesions. The lesion-related mRNA expression and secretion of VEGF-A and PGF by mononuclear phagocytes were potently suppressed by PGF and partially by VEGF-A inhibition. Protein levels of IL-6 and IL1-beta were strongly reduced in all treatment groups. CONCLUSIONS: Retinal inhibition of PGF in combination with VEGF-A prevents vascular leakage and CNV possibly via modulating their own expression in mononuclear phagocytes. PGF-related, optimized strategies to target inflammation-mediated angiogenesis may help to increase efficacy and reduce non-responders in the treatment of wet AMD patients.

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

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.

NADPH Oxidase 3: Beyond the Inner Ear.

Reactive oxygen species (ROS) were formerly known as mere byproducts of metabolism with damaging effects on cellular structures. The discovery and description of NADPH oxidases (Nox) as a whole enzyme family that only produce this harmful group of molecules was surprising. After intensive research, seven Nox isoforms were discovered, described and extensively studied. Among them, the NADPH oxidase 3 is the perhaps most underrated Nox isoform, since it was firstly discovered in the inner ear. This stigma of Nox3 as "being only expressed in the inner ear" was also used by me several times. Therefore, the question arose whether this sentence is still valid or even usable. To this end, this review solely focuses on Nox3 and summarizes its discovery, the structural components, the activating and regulating factors, the expression in cells, tissues and organs, as well as the beneficial and detrimental effects of Nox3-mediated ROS production on body functions. Furthermore, the involvement of Nox3-derived ROS in diseases progression and, accordingly, as a potential target for disease treatment, will be discussed.

Encapsulation of propolis extracted with methylal in the chitosan nanoparticles and its antibacterial and cell cytotoxicity studies.

In this study we develop novel type of antibacterial chitosan-propolis NPs to improve theantimicrobial activity against various pathogens. To this aim, we primarily extracted propolis with methylal and ethanol as green solvents and its encapsulation with chitosan NPs. The developed propolis loaded chitosan NPs indicated antimicrobial and anti-biofilm properties against various gram positive and negative. FTIR revealed the successful encapsulation of the propolis extract with Ethanol (PE) and Methylal (PM) into the chitosan nano career matrix. HPLC and GC-MASS also confirmed the presence of flavonoids and phenols compounds of propolis extracted with both solvents. In addition, we confirmed the total phenolic and flavonoid compounds in propolis by calorimetric method of Folin-Ciocalteu and aluminum trichloride complex formation assays, respectively. PE-CH and PM-CH were optimized regarding physicochemical properties such as particle size, zeta potential, and poly dispersity index (PDI) index. DLS and SEM micrographs confirmed a spherical morphology in a range of 360-420 nm with Z potential values of 30-48 mV and PDI of 0.105-0.166 for PE-CH and PM-CH, respectively. The encapsulation efficiency was evaluated using colorimetric analysis, with median values ranging from 90 to 92%. The MIC values within the range of 2 to 230 µg/ml and MBC values between 3 to 346 µg/ml against both gram-positive and negative bacteria. While both PE and PM showed a significant reduction in the number of E. coli, S. aureus, and S. epidermidis, the use of PE-CH and PM-CH led to a statistically significant and greater reduction in number of E. coli, S. aureus, and S. epidermidis strains on the biofilm, pre-formed biofilm and planktonic phases. Besides, the DPPH assay showed significant antioxidant activity for these NPs within the range of 36 to 92%. MTT assay for MHFB-1, HFF, L929, MDF, and MCF-7 cells exhibited statistically significant differences in each other that show the IC50 between 60-160 µg/ml for normal cells and 20 for cancer cells. Finally the present study indicated that both PM and PM-CH greater than PE and PE-CH in which contain high flavonoid and phenolic contents with a high antioxidation potential antioxidant properties, which could be beneficial for cell proliferation and antibiotic and anticancer applications.

3D Printing of Biocompatible and Antibacterial Silica-Silk-Chitosan-Based Hybrid Aerogel Scaffolds Loaded with Propolis.

The aim of this study is to design a therapeutic enhanced three-dimensional (3D) silk fibroin (SF)-based scaffold containing propolis (Ps)-loaded chitosan (CH) nanocarriers. To this aim, we initially synthesized a hybrid gel-based ink by a synergistic sol-gel and self-assembly approach and then processed the resulting gels by microextrusion-based 3D printing followed by supercritical drying to obtain 3D hybrid aerogel scaffolds. Ps was utilized to enhance the final scaffold's bactericidal efficacy and cell responsiveness. For the synthesis of the scaffold, two Ps loading methods (in preprint and postprinting steps) were investigated in order to optimize the Ps drug quantities in the scaffold and maximize the antibacterial properties of scaffold. In the postprinting Ps loading step, the hybrid silica-oxidized SF (SFO)-CH hydrogel ink was 3D printed into a construct with an interconnected porous structure, and then, Ps was loaded into the printed construct. In the preprint loading method, PS was incorporated into the SF and a hydrolyzed silane solution prior to gelation. The morphological studies demonstrate that the addition of Ps encapsulated CH nanoparticles (NPs) into the hydrogel solution improved the porosity of the developed scaffolds. The rheological analysis of the designed gel ink with and without Ps loading and the release kinetics were studied. The antimicrobial results show that the Ps-loaded scaffolds in the postprinting step exhibited superior antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) strains compared to a preprinted Ps-loaded scaffold. Direct and indirect in vitro cytotoxicity tests also confirmed the designed Ps-loaded scaffold biocompatibility toward a mouse fibroblast (L929) cell line. We demonstrated that the scaffold formulated by propolis-loaded chitosan NPs can enhance the migration and proliferation of L929 fibroblast cells. The obtained results prove the promise of the designed 3D printed silica-SFO-CH-Ps scaffolds as a potent 3D scaffold to mediate tissue regeneration but also as an antibacterial highly porous matrix to support wound healing.

Designing of a Multifunctional 3D-Printed Biomimetic Theragenerative Aerogel Scaffold via Mussel-Inspired Chemistry: Bioactive Glass Nanofiber-Incorporated Self-Assembled Silk Fibroin with Antibacterial, Antiosteosarcoma, and Osteoinductive Properties.

Biomaterial-mediated bone tissue engineering (BTE) offers an alternative, interesting approach for the restoration of damaged bone tissues in postsurgery osteosarcoma treatment. This study focused on synthesizing innovative composite inks, integrating self-assembled silk fibroin (SF), tannic acids (TA), and electrospun bioactive glass nanofibers 70SiO2-25CaO-5P2O5 (BGNF). By synergistically combining the unique characteristics of these three components through self-assembly and microextrusion-based three-dimensional (3D) printing, our goal was to produce durable and versatile aerogel-based 3D composite scaffolds. These scaffolds were designed to exhibit hierarchical porosity along with antibacterial, antiosteosarcoma, and bone regeneration properties. Taking inspiration from mussel foot protein attachment chemistry involving the coordination of dihydroxyphenylalanine (DOPA) amino acids with ferric ions (Fe3+), we synthesized a tris-complex catecholate-iron self-assembled composite gel. This gel formation occurred through the coordination of oxidized SF (SFO) with TA and polydopamine-modified BGNF (BGNF-PDA). The dynamic nature of the coordination ligand-metal bonds within the self-assembled SFO matrix provided excellent shear-thinning properties, allowing the SFO-TA-BGNF complex gel to be extruded through a nozzle, facilitating 3D printing into scaffolds with outstanding shape fidelity. Moreover, the developed composite aerogels exhibited multifaceted features, including NIR-triggered photothermal antibacterial and in vitro photothermal antiosteosarcoma properties. In vitro studies showcased their excellent biocompatibility and osteogenic features as seeded cells successfully differentiated into osteoblasts, promoting bone regeneration in 21 days. Through comprehensive characterizations and biological validations, our antibacterial scaffold demonstrated promise as an exceptional platform for concurrent bone regeneration and bone cancer therapy, setting the stage for their potential clinical application.

When the Phagosome Gets Leaky: Pore-Forming Toxin-Induced Non-Canonical Autophagy (PINCA).

Macrophages remove bacteria from the extracellular milieu via phagocytosis. While most of the engulfed bacteria are degraded in the antimicrobial environment of the phagolysosome, several bacterial pathogens have evolved virulence factors, which evade degradation or allow escape into the cytosol. To counter this situation, macrophages activate LC3-associated phagocytosis (LAP), a highly bactericidal non-canonical autophagy pathway, which destroys the bacterial pathogens in so called LAPosomes. Moreover, macrophages can also target intracellular bacteria by pore-forming toxin-induced non-canonical autophagy (PINCA), a recently described non-canonical autophagy pathway, which is activated by phagosomal damage induced by bacteria-derived pore-forming toxins. Similar to LAP, PINCA involves LC3 recruitment to the bacteria-containing phagosome independently of the ULK complex, but in contrast to LAP, this process does not require ROS production by Nox2. As last resort of autophagic targeting, macrophages activate xenophagy, a selective form of macroautophagy, to recapture bacteria, which evaded successful targeting by LAP or PINCA through rupture of the phagosome. However, xenophagy can also be hijacked by bacterial pathogens for their benefit or can be completely inhibited resulting in intracellular growth of the bacterial pathogen. In this perspective, we discuss the molecular differences and similarities between LAP, PINCA and xenophagy in macrophages during bacterial infections.

Macrophages target Listeria monocytogenes by two discrete non-canonical autophagy pathways.

Non-canonical autophagy pathways decorate single-membrane vesicles with Atg8-family proteins such as MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). Phagosomes containing the bacterial pathogen Listeria monocytogenes (L.m.) can be targeted by a non-canonical autophagy pathway called LC3-associated phagocytosis (LAP), which substantially contributes to the anti-listerial activity of macrophages and immunity. We here characterized a second non-canonical autophagy pathway targeting L.m.-containing phagosomes, which is induced by damage caused to the phagosomal membrane by the pore-forming toxin of L.m., listeriolysin O. This pore-forming toxin-induced non-canonical autophagy pathway (PINCA) was the only autophagic pathway evoked in tissue macrophages deficient for the NADPH oxidase CYBB/NOX2 that produces the reactive oxygen species (ROS) that are required for LAP induction. Similarly, also bone marrow-derived macrophages (BMDM) exclusively targeted L.m. by PINCA as they completely failed to induce LAP because of insufficient production of ROS through CYBB, in part, due to low expression of some CYBB complex subunits. Priming of BMDM with proinflammatory cytokines such as TNF and IFNG/IFNγ increased ROS production by CYBB and endowed them with the ability to target L.m. by LAP. Targeting of L.m. by LAP remained relatively rare, though, preventing LAP from substantially contributing to the anti-listerial activity of BMDM. Similar to LAP, the targeting of L.m.-containing phagosomes by PINCA promoted their fusion with lysosomes. Surprisingly, however, this did not substantially contribute to anti-listerial activity of BMDM. Thus, in contrast to LAP, PINCA does not have clear anti-listerial function suggesting that the two different non-canonical autophagy pathways targeting L.m. may have discrete functions.Abbreviations: actA/ActA: actin assembly-inducing protein A; ATG: autophagy-related; BMDM: Bone marrow-derived macrophages; CALCOCO2/NDP52: calcium-binding and coiled-coil domain-containing protein 2; CYBA/p22phox: cytochrome b-245 light chain; CYBB/NOX2: cytochrome b(558) subunit beta; E. coli: Escherichia coli; IFNG/IFNγ: interferon gamma; L.m.: Listeria monocytogenes; LAP: LC3-associated phagocytosis; LGALS: galectin; LLO: listeriolysin O; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NCF1/p47phox: neutrophil cytosol factor 1; NCF2/p67phox: neutrophil cytosol factor 2; NCF4/p67phox: neutrophil cytosol factor 4; Peritoneal macrophages: PM; PINCA: pore-forming toxin-induced non-canonical autophagy; plc/PLC: 1-phosphatidylinositol phosphodiesterase; PMA: phorbol 12-myristate 13-acetate; RB1CC1/FIP200: RB1-inducible coiled-coil protein 1; ROS: reactive oxygen species; S. aureus: Staphylococcus aureus; S. flexneri: Shigella flexneri; SQSTM1/p62: sequestosome 1; S. typhimurium: Salmonella typhimurium; T3SS: type III secretion system; TNF: tumor necrosis factor; ULK: unc-51 like autophagy activating kinase; PM: peritoneal macrophages; WT: wild type.

Fabrication of Antibacterial, Osteo-Inductor 3D Printed Aerogel-Based Scaffolds by Incorporation of Drug Laden Hollow Mesoporous Silica Microparticles into the Self-Assembled Silk Fibroin Biopolymer.

In this study, the novel biomimetic aerogel-based composite scaffolds through a synergistic combination of wet chemical synthesis and advanced engineering approaches have successfully designed. To this aim, initially the photo-crosslinkable methacrylated silk fibroin (SF-MA) biopolymer and methacrylated hollow mesoporous silica microcapsules (HMSC-MA) as the main constituents of the novel composite aerogels were synthesized. Afterward, by incorporation of drug-loaded HMSC-MA into the self-assembled SF-MA, printable gel-based composite inks are developed. By exploiting micro-extrusion-based three-dimensional (3D) printing, SF-MA-HMSC composite gels are printed by careful controlling their viscosity to provide a means to control the shape fidelity of the resulted printed gel constructs. The developed scaffold has shown a multitude of interesting biophysical and biological performances. Namely, thanks to the photo-crosslinking of the gel components during the 3D printing, the scaffolds become mechanically more stable than the pristine SF scaffolds. Also, freeze-casting the printed constructs generates further interconnectivity in the printed pore struts resulting in the scaffolds with hierarchically organized porosities necessary for cell infiltration and growth. Importantly, HMSC incorporated scaffolds promote antibacterial drug delivery, cellular ingrowth and proliferation, promoting osteoblastic differentiation by inducing the expression of osteogenic markers and matrix mineralization. Finally, the osteoconductive, -inductive, and anti-infective composite aerogels are expected to act as excellent bone implanting materials with an extra feature of local and sustained release of drug for efficient therapy of bone-related diseases.

Functions of ROS in Macrophages and Antimicrobial Immunity.

Reactive oxygen species (ROS) are a chemically defined group of reactive molecules derived from molecular oxygen. ROS are involved in a plethora of processes in cells in all domains of life, ranging from bacteria, plants and animals, including humans. The importance of ROS for macrophage-mediated immunity is unquestioned. Their functions comprise direct antimicrobial activity against bacteria and parasites as well as redox-regulation of immune signaling and induction of inflammasome activation. However, only a few studies have performed in-depth ROS analyses and even fewer have identified the precise redox-regulated target molecules. In this review, we will give a brief introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense, and provide an overview of commonly used ROS probes, scavengers and inhibitors.

Reactive Oxygen Species: Not Omnipresent but Important in Many Locations.

Reactive oxygen species (ROS), such as the superoxide anion or hydrogen peroxide, have been established over decades of research as, on the one hand, important and versatile molecules involved in a plethora of homeostatic processes and, on the other hand, as inducers of damage, pathologies and diseases. Which effects ROS induce, strongly depends on the cell type and the source, amount, duration and location of ROS production. Similar to cellular pH and calcium levels, which are both strictly regulated and only altered by the cell when necessary, the redox balance of the cell is also tightly regulated, not only on the level of the whole cell but in every cellular compartment. However, a still widespread view present in the scientific community is that the location of ROS production is of no major importance and that ROS randomly diffuse from their cellular source of production throughout the whole cell and hit their redox-sensitive targets when passing by. Yet, evidence is growing that cells regulate ROS production and therefore their redox balance by strictly controlling ROS source activation as well as localization, amount and duration of ROS production. Hopefully, future studies in the field of redox biology will consider these factors and analyze cellular ROS more specifically in order to revise the view of ROS as freely flowing through the cell.

NOX2 Deficiency Permits Sustained Survival of S. aureus in Macrophages and Contributes to Severity of Infection.

Although the crucial role of professional phagocytes for the clearance of S. aureus infections is well-established, several studies indicate an adverse role of leukocytes in the dissemination of S. aureus during infection. Since only little is known about macrophages in this context, we analyzed the role of macrophages, and in particular reactive oxygen species deficiency, for the seeding of S. aureus metastases. Infection of bone marrow-derived macrophages (BMDM) with S. aureus revealed that NADPH oxidase 2 (NOX2-) deficient, but not NOX1- or NOX4-deficient, BMDM failed to clear intracellular S. aureus. Despite of larger intracellular bacterial burden, NOX2-deficient BMDM showed significantly improved survival. Intravenous injection of mice with in vitro-infected BMDMs carrying intracellular viable S. aureus led to higher bacterial loads in kidney and liver of mice compared to injection with plain S. aureus. An even higher frequency of liver abscesses was observed in mice infected with S. aureus-loaded nox2-/- BMDM. Thus, the improved intracellular survival of S. aureus and improved viability of NOX2-deficient BMDM is associated with an aggravated metastatic dissemination of S. aureus infection. A combination of vancomycin and the intracellularly active antibiotic rifampicin led to complete elimination of S. aureus from liver within 48 h, which was not achieved with vancomycin treatment alone, underscoring the impact of intracellular S. aureus on the course of disease. The results of our study indicate that intracellular S. aureus carried by macrophages are sufficient to establish a systemic infection. This suggests the inclusion of intracellularly active antibiotics in the therapeutic regimen of invasive S. aureus infections, especially in patients with NADPH oxidase deficiencies such as chronic granulomatous disease.

The TSPO-NOX1 axis controls phagocyte-triggered pathological angiogenesis in the eye.

Aberrant immune responses including reactive phagocytes are implicated in the etiology of age-related macular degeneration (AMD), a major cause of blindness in the elderly. The translocator protein (18 kDa) (TSPO) is described as a biomarker for reactive gliosis, but its biological functions in retinal diseases remain elusive. Here, we report that tamoxifen-induced conditional deletion of TSPO in resident microglia using Cx3cr1CreERT2:TSPOfl/fl mice or targeting the protein with the synthetic ligand XBD173 prevents reactivity of phagocytes in the laser-induced mouse model of neovascular AMD. Concomitantly, the subsequent neoangiogenesis and vascular leakage are prevented by TSPO knockout or XBD173 treatment. Using different NADPH oxidase-deficient mice, we show that TSPO is a key regulator of NOX1-dependent neurotoxic ROS production in the retina. These data define a distinct role for TSPO in retinal phagocyte reactivity and highlight the protein as a drug target for immunomodulatory and antioxidant therapies for AMD.

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 recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

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

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 Listeria monocytogenes-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 Cys54 and Cys347, 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.

Corticosteroids inhibit Mycobacterium tuberculosis-induced necrotic host cell death by abrogating mitochondrial membrane permeability transition.

Corticosteroids are host-directed drugs with proven beneficial effect on survival of tuberculosis (TB) patients, but their precise mechanisms of action in this disease remain largely unknown. Here we show that corticosteroids such as dexamethasone inhibit necrotic cell death of cells infected with Mycobacterium tuberculosis (Mtb) by facilitating mitogen-activated protein kinase phosphatase 1 (MKP-1)-dependent dephosphorylation of p38 MAPK. Characterization of infected mixed lineage kinase domain-like (MLKL) and tumor necrosis factor receptor 1 (TNFR1) knockout cells show that the underlying mechanism is independent from TNFα-signaling and necroptosis. Our results link corticosteroid function and p38 MAPK inhibition to abrogation of necrotic cell death mediated by mitochondrial membrane permeability transition, and open new avenues for research on novel host-directed therapies (HDT).

LC3-associated phagocytosis initiated by integrin ITGAM-ITGB2/Mac-1 enhances immunity to 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 αMß2 as the receptor that initiates LAP in response to Listeria infection.

The ß2 Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes.

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 ß2 integrin Mac-1 (CR3, integrin αMß2), 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.