Host and Bacterial Proteins That Repress Recruitment of LC3 to Shigella Early during Infection

Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min.), the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4–6 hr) by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG). Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection

The Contribution of Melanoregulin to Microtubule-Associated Protein 1 Light Chain 3 (LC3) Associated Phagocytosis in Retinal Pigment Epithelium

A main requisite in the phagocytosis of ingestedmaterial is a coordinated series of maturation steps which leadto the degradation of ingested cargo. Photoreceptor outersegment (POS) renewal involves phagocytosis of the distaldisk membranes by the retinal pigment epithelium (RPE).Previously, we identified melanoregulin (MREG) as an intra-cellular cargo-sorting protein required for the degradation ofPOS disks. Here, we provide evidence that MREG-dependentprocessing links both autophagic and phagocytic processes inLC3-associated phagocytosis (LAP). Ingested POSphagosomes are associated with endogenous LC3 andMREG. The LC3 association with POSs exhibited propertiesof LAP; it was independent of rapamycin pretreatment, butdependent on Atg5. Loss of MREG resulted in a decrease inthe extent of LC3-POS association. Studies using DQ™-BSAsuggest that loss of MREG does not compromise the associ-ation and fusion of LC3-positive phagosomes with lysosomes.Furthermore, the mechanism of MREG action is likelythrough a protein complex that includes LC3, as determinedby colocalization and immunoprecipitation in both RPE cellsand macrophages. We posit that MREG participates incoordinating the association of phagosomes with LC3 forcontent degradation with the loss of MREG leading tophagosome accumulation

Interplay between FGFR2b-induced autophagy and phagocytosis: role of PLCγ-mediated signalling

Signalling of the epithelial splicing variant of the fibroblast growth factor receptor 2 (FGFR2b) induces both autophagy and phagocytosis in human keratinocytes. Here, we investigated, in the cell model of HaCaT keratinocytes, whether the two processes might be related and the possible involvement of PLCγ signalling. Using fluorescence and electron microscopy, we demonstrated that the FGFR2b-induced phagocytosis and autophagy involve converging autophagosomal and phagosomal compartments. Moreover, the forced expression of FGFR2b signalling mutants and the use of specific inhibitors of FGFR2b substrates showed that the receptor-triggered autophagy requires PLCγ signalling, which in turn activates JNK1 via PKCδ. Finally, we found that in primary human keratinocytes derived from light or dark pigmented skin and expressing different levels of FGFR2b, the rate of phagocytosis and autophagy and the convergence of the two intracellular pathways are dependent on the level of receptor expression, suggesting that FGFR2b signalling would control in vivo the number of melanosomes in keratinocytes, determining skin pigmentation

Differential Kinetics of Aspergillus nidulans and Aspergillus fumigatus Phagocytosis

Acknowledgements: The authors would like to acknowledge Fraser P. Coxon and Ian Ganley for providing LC3-GFP-mCherry BMDMs. M.S.G. was supported by an FEMS research grant and F.L.v.d.V. was supported by ZonMW under the name EURO-CMC frame of E-Rare-2, the ERA-Net for Research on Rare Diseases.Peer reviewedPublisher PD

The Contribution of Melanoregulin to Microtubule-Associated Protein 1 Light Chain 3 (LC3) Associated Phagocytosis in Retinal Pigment Epithelium

A main requisite in the phagocytosis of ingested material is a coordinated series of maturation steps which lead to the degradation of ingested cargo. Photoreceptor outer segment (POS) renewal involves phagocytosis of the distal disk membranes by the retinal pigment epithelium (RPE). Previously, we identified melanoregulin (MREG) as an intracellular cargo-sorting protein required for the degradation of POS disks. Here, we provide evidence that MREG-dependent processing links both autophagic and phagocytic processes in LC3-associated phagocytosis (LAP). Ingested POS phagosomes are associated with endogenous LC3 and MREG. The LC3 association with POSs exhibited properties of LAP; it was independent of rapamycin pretreatment, but dependent on Atg5. Loss of MREG resulted in a decrease in the extent of LC3-POS association. Studies using DQ™-BSA suggest that loss of MREG does not compromise the association and fusion of LC3-positive phagosomes with lysosomes. Furthermore, the mechanism of MREG action is likely through a protein complex that includes LC3, as determined by colocalization and immunoprecipitation in both RPE cells and macrophages. We posit that MREG participates in coordinating the association of phagosomes with LC3 for content degradation with the loss of MREG leading to phagosome accumulation. © 2014, Springer Science+Business Media New York

The interrelationship between phagocytosis, autophagy and formation of neutrophil extracellular traps following infection of human neutrophils by Streptococcus pneumoniae

Neutrophils play an important role in the innate immune response to infection with Streptococcus pneumoniae, the pneumococcus. Pneumococci are phagocytosed by neutrophils and undergo killing after ingestion. Other cellular processes may also be induced, including autophagy and the formation of neutrophil extracellular traps (NETs), which may play a role in bacterial eradication. We set out to determine how these different processes interacted following pneumococcal infection of neutrophils, and the role of the major pneumococcal toxin pneumolysin in these various pathways. We found that pneumococci induced autophagy in neutrophils in a type III phosphatidylinositol-3 kinase dependent fashion that also required the autophagy gene Atg5. Pneumolysin did not affect this process. Phagocytosis was inhibited by pneumolysin but enhanced by autophagy, while killing was accelerated by pneumolysin but inhibited by autophagy. Pneumococci induced extensive NET formation in neutrophils that was not influenced by pneumolysin but was critically dependent on autophagy. While pneumolysin did not affect NET formation, it had a potent inhibitory effect on bacterial trapping within NETs. These findings show a complex interaction between phagocytosis, killing, autophagy and NET formation in neutrophils following pneumococcal infection that contribute to host defence against this pathogen

Caspase-1 cleavage of the TLR adaptor TRIF inhibits autophagy and β-interferon production during pseudomonas aeruginosa infection

Bacterial infection can trigger autophagy and inflammasome activation, but the effects of inflammasome activation on autophagy are unknown. We examined this in the context of Pseudomonas aeruginosa macrophage infection, which triggers NLRC4 inflammasome activation. P. aeruginosa induced autophagy via TLR4 and its adaptor TRIF. NLRC4 and caspase-1 activation following infection attenuated autophagy. Caspase-1 directly cleaved TRIF to diminish TRIF-mediated signaling, resulting in inhibition of autophagy and in reduced type I interferon production. Expression of a caspase-1 resistant TRIF mutant enhanced autophagy and type I interferon production following infection. Preventing TRIF cleavage by caspase-1 in an in vivo model of P. aeruginosa infection resulted in enhanced bacterial autophagy, attenuated IL-1β production, and increased bacterial clearance. Additionally, TRIF cleavage by caspase-1 diminished NLRP3 inflammasome activation. Thus, caspase-1 mediated TRIF cleavage is a key event in controlling autophagy, type I interferon production, and inflammasome activation with important functional consequences

ATG proteins mediate efferocytosis and suppress inflammation in mammary involution.

Involution is the process of post-lactational mammary gland regression to quiescence and it involves secretory epithelial cell death, stroma remodeling and gland repopulation by adipocytes. Though reportedly accompanying apoptosis, the role of autophagy in involution has not yet been determined. We now report that autophagy-related (ATG) proteins mediate dead cell clearance and suppress inflammation during mammary involution. In vivo, Becn1(+/-) and Atg7-deficient mammary epithelial cells (MECs) produced 'competent' apoptotic bodies, but were defective phagocytes in association with reduced expression of the MERTK and ITGB5 receptors, thus pointing to defective apoptotic body engulfment. Atg-deficient tissues exhibited higher levels of involution-associated inflammation, which could be indicative of a tumor-modulating microenvironment, and developed ductal ectasia, a manifestation of deregulated post-involution gland remodeling. In vitro, ATG (BECN1 or ATG7) knockdown compromised MEC-mediated apoptotic body clearance in association with decreased RAC1 activation, thus confirming that, in addition to the defective phagocytic processing reported by other studies, ATG protein defects also impair dead cell engulfment. Using two different mouse models with mammary gland-associated Atg deficiencies, our studies shed light on the essential role of ATG proteins in MEC-mediated efferocytosis during mammary involution and provide novel insights into this important developmental process. This work also raises the possibility that a regulatory feedback loop exists, by which the efficacy of phagocytic cargo processing in turn regulates the rate of engulfment and ultimately determines the kinetics of phagocytosis and dead cell clearance

CD5L promotes M2 macrophage polarization through autophagy-mediated upregulation of ID3

CD5L (CD5 molecule-like) is a secreted glycoprotein that controls key mechanisms in inflammatory responses, with involvement in processes such as infection, atherosclerosis, and cancer. In macrophages, CD5L promotes an anti-inflammatory cytokine profile in response to TLR activation. In the present study, we questioned whether CD5L is able to influence human macrophage plasticity, and drive its polarization toward any specific phenotype. We compared CD5L-induced phenotypic and functional changes to those caused by IFN/LPS, IL4, and IL10 in human monocytes. Phenotypic markers were quantified by RT-qPCR and flow cytometry, and a mathematical algorithm was built for their analysis. Moreover, we compared ROS production, phagocytic capacity, and inflammatory responses to LPS. CD5L drove cells toward a polarization similar to that induced by IL10. Furthermore, IL10- and CD5L-treated macrophages showed increased LC3-II content and colocalization with acidic compartments, thereby pointing to the enhancement of autophagy-dependent processes. Accordingly, siRNA targeting ATG7 in THP1 cells blocked CD5L-induced CD163 and Mer tyrosine kinase mRNA and efferocytosis. In these cells, gene expression profiling and validation indicated the upregulation of the transcription factor ID3 by CD5L through ATG7. In agreement, ID3 silencing reversed polarization by CD5L. Our data point to a significant contribution of CD5L-mediated autophagy to the induction of ID3 and provide the first evidence that CD5L drives macrophage polarization.Peer ReviewedPostprint (published version

Autophagy in Microglia and Alzheimer's disease

Alzheimer’s disease (AD) is the most common neurodegenerative disease, characterized by amyloid-beta plaques, neurofibrillary tangles and neuroinflammation. Autophagy has been associated with several neurodegenerative diseases. Recently, autophagy has been linked to the regulation of the inflammatory response in macrophages. My thesis investigates how an impairment of autophagy influences the inflammatory response of microglia. We used Beclin1 heterozygous (Becn1+/-) mice as a model of impaired autophagy. Beclin1 plays a role in the initiation of autophagy and was shown to be decreased in microglia isolated from AD patients compared to healthy controls. In vitro, acutely stimulated microglia from neonatal Becn1+/- mice exhibited increased expression of the proinflammatory cytokines IL-1beta and IL-18 compared to wild type microglia. Both IL-1beta and IL-18 are processed by the NLRP3 inflammasome pathway. The investigation of this pathway showed an elevated number of cells with inflammasomes and increased levels of the inflammasome components NLRP3 and cleaved Caspase1 in Becn1+/- microglia. Super resolution microscopy revealed a very close association of NLRP3 aggregates and LC3-positive autophagosomes. Interestingly, despite suggestions that the murine CALCOCO2 does not function as an autophagic adaptor, we discovered CALCOCO2 colocalised with NLRP3 and that its downregulation by siRNA knockdown increased IL-1beta release. These data support the notion that selective autophagy can impact microglia activation by modulating IL-1beta and IL-18 production via NLRP3 degradation. These in vitro data present a mechanism how impaired autophagy could contribute to neuroinflammation in AD. In vivo analysis of Becn1+/-.APPPS1 mice also demonstrated enhanced IL-1beta levels, but no differences in amyloid beta pathology, nor phagocytic capacity. The constitutive heterozygosity of Beclin1 might be responsible for the milder effects in vivo. Therefore, we performed studies utilizing more sophisticated models targeting immune cells specifically. The first model, Aldh1l1-iCre.Becn1-flox, targets Becn1 deletion specifically in astrocytes in the central nervous system after injection with the drug tamoxifen. Peripherally, Aldh1l1 is also expressed by hepatocytes. The Aldh1l1-iCre.Becn1-flox mice suffered from peripheral damage in the liver 10 days after tamoxifen injection, and can therefore not be used in further studies. The second model, Cx3Cr1-iCre.Becn1-flox, targets Becn1 deletion specifically in microglia in the central nervous system, and will be crossed to the APPPS1 mice to create a tool to study the role of Beclin1 in microglia in neuroinflammation and neurodegeneration. This new tool and the data generated in this work will support a new direction of research, to unravel the therapeutic potential of autophagy-dependent inflammation in neurodegenerative diseases.Die Alzheimer-Krankheit (AD) ist die häufigste neurodegenerative Erkrankung, die durch Amyloid-Beta-Plaques, neurofibrilläre Verwicklungen und Neuroinflammation gekennzeichnet ist. Autophagie wurde mit mehreren neurodegenerativen Erkrankungen in Verbindung gebracht. Vor Kurzem wurde Autophagie mit der Regulierung der Entzündungsreaktion in Makrophagen in Verbindung gebracht. Meine Dissertation untersucht, wie eine Beeinträchtigung der Autophagie die Entzündungsreaktion von Mikroglia beeinflusst. Wir haben Beclin1-heterozygote (Becn1+/-) Mäuse als Modell für eingeschränkte Autophagie verwendet. Beclin1 spielt eine Rolle bei der Initiierung der Autophagie und es wurde gezeigt, dass es bei aus AD-Patienten isolierten Mikrogliazellen im Vergleich zu gesunden Kontrollen abnimmt. Akut stimulierte Mikroglia aus neonatalen Becn1+/– Mäusen zeigten in vitro eine erhöhte Expression der proinflammatorischen Zytokine IL-1beta und IL-18 im Vergleich zu Wildtyp-Mikroglia. Sowohl IL-1beta als auch IL-18 werden vom NLRP3-Inflammasom-Weg verarbeitet. Die Untersuchung dieses Weges zeigte eine erhöhte Anzahl von Zellen mit Inflammasomen und erhöhte Spiegel der Inflammasomenkomponenten NLRP3 und gespaltenen Caspase1 in Becn1+/– Mikroglia. Super-Resolution-Mikroskopie zeigte eine sehr enge Lokalisation von NLRP3-Aggregaten und LC3-positiven Autophagosomen. Interessanterweise haben wir trotz der Kritik, dass das murine CALCOCO2 nicht als autophagischer Adapter fungiert, entdeckt, dass CALCOCO2 mit NLRP3 kolokalisiert und dass die Herunterregulierung durch siRNA die IL-1beta-Freisetzung erhöhte. Diese Daten stützen die Ansicht, dass selektive Autophagie die Mikroglia-Aktivierung beeinflussen kann, indem die IL-1beta- und IL-18-Produktion durch NLRP3-Abbau moduliert wird. Diese in vitro Daten stellen einen Mechanismus dar, wie eine gestörte Autophagie zur Neuroinflammation bei AD beitragen kann. In vivo Analysen von Becn1+/–.APPPS1 Mäusen zeigten ebenfalls erhöhte IL-1beta-Spiegel, jedoch keine Unterschiede in der Amyloid-Beta-Pathologie und auch keine in Bezug auf die Phagozytosekapazität. Die konstitutive Heterozygotie von Beclin1 könnte für die geringen Auswirkungen in vivo verantwortlich sein. Daher etablierten zwei neue Modelle, die speziell auf Immunzellen abzielten. Das erste Modell, Aldh1l1-iCre.Becn1-Flox, zielt auf die Becn1-Deletion spezifisch in Astrozyten im zentralen Nervensystem nach Injektion des Arzneimittels Tamoxifen ab. In der Peripherie wird Aldh1l1 auch von Hepatozyten exprimiert. Die Aldh1l1-iCre.Becn1-Flox Mäuse erlitten 10 Tage nach Tamoxifen-Injektion eine periphere Schädigung der Leber und können daher nicht in weiteren Studien verwendet werden. Das zweite Modell, Cx3Cr1-iCre.Becn1-flox, zielt auf die Becn1-Deletion speziell in Mikroglia im Zentralnervensystem ab und wird mit den APPPS1-Mäusen gekreuzt, um ein Modell für die Untersuchung der Rolle von Beclin1 in Mikroglia bei Neuroinflammation und Neurodegeneration darzustellen. Dieses neue Mausmodell und die in dieser Arbeit generierten Daten werden eine neue Richtung der Forschung unterstützen, um das therapeutische Potenzial autophagieabhängiger Entzündungen bei neurodegenerativen Erkrankungen zu ermitteln