Annemarie H. Meijer

BioStudies – one package for all the data supporting a study

Glucocorticoids are widely prescribed anti-inflammatory drugs that act on nearly all cell types due to the ubiquitous expression of the glucocorticoid receptor (GR). While the molecular mechanisms of GR activation have been studied extensively, it is less well understood how GR signaling modulates cell type-specific responses during inflammation. Using the unique attributes of the zebrafish, we performed single-cell RNA sequencing to characterize GR-mediated transcriptional regulation under conditions of wounding-induced inflammation. We found specific cell populations that occurred only under inflammatory conditions within epidermal cells, macrophages and neutrophils. The inflammation-specific subset of epidermal cells showed elevated expression of inflammatory markers, and these cells exhibited a reduced transactivation response to dexamethasone. This reduction in GR activation was also observed in the inflammation-specific subset of macrophages, but not in neutrophils, which retained dexamethasone responsiveness. By correlating the dexamethasone responsiveness with transcriptional coregulator expression, we identified candidate regulatory mechanisms underlying differential GR responsiveness across cell types. Taken together, we provided unique mechanistic insights into cell differentiation during a local inflammatory response and cell type-specific glucocorticoid responsiveness in vivo .

Guanylate-binding proteins (GBP) are a family of interferon-inducible GTPases that operate via cell-autonomous immunity against intracellular pathogens and regulate cell death responses of infected host cells. GBPs have emerged as robust markers of human mycobacterial diseases, including tuberculosis and leprosy. However, their function in mycobacterial infections remains poorly understood. Here, using a zebrafish infection model with Mycobacterium marinum , a pathogen frequently used to model tuberculosis, we report that members of the GBP family differentially regulate both the host defense ability and the extent of cell death induced by mycobacterial infection. By genetically inhibiting each of the four zebrafish GBPs, we show that three of the family members (Gbp2, Gbp3 and Gbp4) contribute to host resistance. Strikingly, zebrafish larvae showed an improved ability to restrict mycobacterial growth when gbp1 was inhibited, indicating that Gbp1 plays a detrimental role in the response to mycobacterial infection. This effect of Gbp1 was dependent on M. marinum exerting its ESX-1-driven virulence. While none of the Gbp deficiencies affected leukocyte numbers or pathogen acidification, we found that macrophage cell death profiles were altered. Mechanistically, Gbp1 promotes pyroptotic cell death by activating Caspase a in macrophages, thereby attracting neutrophils and increasing mycobacterial dissemination. Additionally, the effect of Gbp1 on pyroptosis correlates with altered tnfa expression in macrophages polarization, as genetic inhibition induced increased expression of this proinflammatory marker. Taken together, our results suggest that the adverse effect of Gbp1 on host resistance to mycobacteria is driven by altered macrophage inflammatory and pyroptotic cell death responses, exacerbating infection. Furthermore, these findings suggest that GBP modulation might be explored as a therapeutic strategy for mycobacterial infections.

Mycobacterium avium , a slow-growing nontuberculous mycobacterium (NTM), is the main cause of life-threatening NTM infections, which are globally on the rise. Unlike Mycobacterium tuberculosis and Mycobacterium marinum , M. avium lacks ESX-1, a subtype of Type VII secretion system (T7SS) and a key virulence determinant. The absence of ESX-1 in M. avium raises questions about its alternative intracellular survival strategies. To investigate M. avium pathogenesis, we exploited our recently established infection model in zebrafish larvae, enabling live imaging of early host-pathogen interactions. Macrophage depletion significantly increased M. avium burden and larval mortality, while neutrophil depletion had no major effect, emphasizing macrophages as key defenders against M. avium . In support, imaging of tnfa activation showed that macrophages polarized to a proinflammatory phenotype. However, like M. marinum , M. avium exploits chemokine receptor Cxcr3.2 signaling in macrophages for its expansion in granuloma-like clusters. Both M. avium and M. marinum preferentially infected macrophages, but M. avium -induced granuloma-like clusters were more compact and exhibited less cell death. Supporting this, lytic cell death pathways were enriched in M. marinum but not M. avium transcriptome signatures. Consequently, we investigated pyroptosis, an important form of inflammation-induced lytic cell death. We found that knockdown of critical mediators of pyroptosis, namely inflammatory caspase a ( caspa ) and gasdermin Eb ( gsdmeb ), produced opposing effects on the two mycobacterial pathogens, indicating a host-protective role during M. avium infection, while exacerbating M. marinum growth. These findings highlight the interaction with host cell death signaling as a determining factor for the pathogenic potential of mycobacterial species.

Dynamic interactions between autophagosomes and lysosomes are key to ensuring the trafficking of mycobacteria through the degradative autophagolysosomal pathway. Phosphoinositides (PtdIns), play critical roles in directing the trafficking of pathogens through phagosomal or autophagosomal degradation pathways. Phosphoinositide kinase, FYVE-type zinc finger containing, PIKfyve catalyzes the phosphorylation of PtdIns(3)P to PtdIns(3,5)P ₂ . Generation of PtdIns(3,5)P ₂ is critical for the maturation of autophagosomes and the proper functioning of lysosomes. The involvement of PIKfyve in immune defense against mycobacterial infections has not been addressed. Here, we studied the role of PIKfyve in Mycobacterium marinum ( Mm ) infection of zebrafish larvae using two chemical inhibitors of the enzymatic activity of PIKfyve, YM201636 and Apilimod. We demonstrate that PIKfyve is required for Mm -containing vesicle trafficking towards degradative lysosomes. We found that in the infected macrophages, PIKfyve facilitates autophagy pathway activation and (auto)phagosome maturation, and protects against cell death, thus boosting the host immune response against mycobacterial infection. This study links PIKfyve to the autolysosomal defense against a mycobacterial pathogen.

Mycobacteria are ancient types of bacteria that can cause serious diseases in both people and animals. The best-known are Mycobacterium tuberculosis, which causes a lung disease called tuberculosis, and Mycobacterium leprae, which causes a disease of the skin and nerves called leprosy. Mycobacteria can spread through the air, through contaminated food or water, or on dirty surfaces. Mycobacteria have clever ways to avoid the body’s defenses, so it is very hard to treat these infections. Patients must take strong medicines called antibiotics for many months. But some mycobacteria have become resistant, meaning they are no longer killed by antibiotics. In this article, we explain what scientists have learned about the tricks mycobacteria use to escape from the body’s defenses and how they cause disease. With this knowledge, scientists hope to find new treatments so that infections with antibiotic-resistant mycobacteria can be cured in the future.

Streptococcus pneumoniae is an opportunistic pathogen responsible for life-threatening diseases including pneumonia and meningitis. The host defense against pneumococci relies heavily on macrophages, which can effectively internalize and degrade bacteria. Recent studies have implicated both canonical and non-canonical autophagy-related processes in bacterial clearance, but the precise pathways mediating defense against S. pneumoniae remain unknown. Here, we utilize a well-established zebrafish larval infection model to investigate the role of autophagy in host defense against pneumococci in vivo. Using a transgenic macroautophagy/autophagy reporter line, we found the autophagy marker Map1lc3/Lc3 being recruited to pneumococci-containing vesicles upon bacterial internalization by zebrafish macrophages. The genetic inhibition of core autophagy gene atg5 led to loss of the Lc3 associations and their impaired acidification, significantly delaying bacterial clearance. This Lc3 recruitment is partially mediated by LC3-associated phagocytosis (LAP), as knockdown of cyba and rubcn moderately reduced Lc3 association with phagosomes and diminished pneumococcal degradation. Interestingly, we observed no involvement of xenophagy components in S. pneumoniae-infected macrophages, suggesting the activation of another non-canonical autophagy pathway, distinct from LAP, targeting pneumococci-containing phagosomes. Instead, we found that the pneumococcal pore-forming toxin pneumolysin induces ROS-independent CASM pathways, one of which is abolished by knockdown of tecpr1a indicating the involvement of sphingomyelin-Tecpr1-induced LC3 lipidation (STIL). Collectively, our observations shed new light on the host immune response against S. pneumoniae, demonstrating that several distinct non-canonical autophagy pathways mediate bacterial degradation by macrophages and providing potential targets for the development of novel therapies to combat pneumococcal infections.Abbreviations: ATG: autophagy related; BMDM: bone marrow-derived macrophage; CASM: conjugation of ATG8 to single membranes; CFU: colony-forming units; Cyba: cytochrome b-245, alpha polypeptide; DPI: diphenyleneiodonium, GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; LAP: LC3-associated phagocytosis; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NADPH: nicotinamide adenine dinucleotide phosphate; Optn: optineurin; PINCA: pore-forming toxin-induced non-canonical autophagy; Ply: pneumolysin; ROS: reactive oxygen species; SLR: sequestosome-like receptors; Sqstm1: sequestosome 1; STIL: sphingomyelin-TECPR1-induced LC3 lipidation; Tecpr1: tectonin beta-propeller repeat containing 1.

Outbreaks of cholera pose a major threat to human health. Currently, antibiotics are the most effective treatment against the causative agent, the bacterium Vibrio cholerae . However, the use of antibiotics eventually leads to the emergence of resistant strains, which necessitates the need for alternative approaches. The use of bacteriophages to target the infection by antibiotic resistant bacteria is one promising alternative. While clearance of Vibrio cholerae with the use of phages has been performed on several animal models, none of these models are natural hosts of V. cholerae . Therefore, we set out to investigate the interaction between V. cholerae and bacteriophage ICP1 both in vitro and in vivo in a natural host, the zebrafish model, Danio rerio . To study the interplay between host, bacteria and phages we used a combination of light and ultrastructural imaging techniques, including confocal fluorescence microscopy, serial block face scanning electron microscopy (EM) imaging and cryogenic EM, which allowed us to investigate both the colonization process by V. cholerae and clearance by the ICP1 bacteriophage. In addition, we determined the effects of the microbiome on this treatment by using germ-free, conventionalized and monoassociated zebrafish larvae as a host. Independent of the presence and composition of microbiomes used here, V. cholerae efficiently colonized the larval intestine. Finally, we demonstrate significant in vivo clearance of V. cholerae N16961-dsRED by ICP1, underscoring the role of phage-bacteria dynamics in shaping pathogen colonization within the zebrafish larval host. SIGNIFICANCE: Cholera remains a life-threatening disease that causes recurring outbreaks and significant mortality, particularly in developing and conflict-affected regions. As antimicrobial resistance continues to rise, there is an urgent need to better understand the ecological and microbial dynamics that govern Vibrio cholerae colonization and persistence. This research investigates how V. cholerae interacts with bacteriophages, the host environment, and the resident microbiota within a natural vertebrate host, offering new insights into the factors that influence pathogen clearance and shaping of the gut ecosystem during infection. The powerful combination of serial block-face scanning and cryogenic electron microscopy, fluorescence microscopy, and traditional colony/plaque counting methods revealed previously unobserved aspects of the interplay between host, pathogen, phages, and the microbiome, highlighting phage-driven clearance of V. cholerae during colonization.

Guanylate-binding proteins (GBPs) are a family of interferon-inducible proteins playing diverse roles in the immunity to infections. By docking onto pathogens or pathogen-containing vesicles, GBPs can boost proinflammatory signaling. However, the interplay between GBPs and the phagolysosomal mechanisms mediating pathogen degradation remains largely unclear. Here, we elucidate the key role of GBP1 in driving lysosomal activity against mycobacterial infection in human macrophages. We show that phagosome damage induced by the mycobacterial ESX-1 secretion system triggers calcium release, and thereby the recruitment of GBP1, which resides in lysosomes upon interferon gamma activation. GBP1 targeting of mycobacteria-containing phagosomes leads to the recruitment of other GBPs but does not lead to the activation of membrane repair mechanisms or antimicrobial autophagy. Instead, GBP1 mediates lysosomal recruitment to intracellular mycobacteria, and promotes the maturation of phagolysosomes and their proteolytic activity required for the degradation of the mycobacterial cargo. Collectively, this work highlights the host-protective function of GBP1 in macrophages, where GBP1 acts as a key cellular sensor of mycobacterial infection and promotes the clearance activity of the host lysosomal system.

Streptococcus pneumoniae is an opportunistic pathogen responsible for life-threatening diseases including pneumonia and meningitis. The host defense against pneumococci relies heavily on macrophages, which can effectively internalize and degrade bacteria. Recent studies have implicated both canonical and non-canonical autophagy-related processes in bacterial clearance, but the precise pathways mediating defense against S. pneumoniae remain unknown. Here, we utilize a well-established zebrafish larval infection model to investigate the role of autophagy in host defense against pneumococci in vivo . Using a transgenic macroautophagy/autophagy reporter line, we found the autophagy marker Map1lc3/Lc3 being recruited to pneumococci-containing vesicles upon bacterial internalization by zebrafish macrophages. The genetic inhibition of core autophagy gene atg5 led to loss of the Lc3 associations and their impaired acidification, significantly delaying bacterial clearance. This Lc3 recruitment is partially mediated by LC3-associated phagocytosis (LAP), as knockdown of cyba and rubcn moderately reduced Lc3 association with phagosomes and diminished pneumococcal degradation. Interestingly, we observed no involvement of xenophagy components in S. pneumoniae -infected macrophages, suggesting the activation of another non-canonical autophagy pathway, distinct from LAP, targeting pneumococci-containing phagosomes. Instead, we found that the pneumococcal pore-forming toxin pneumolysin induces ROS-independent CASM pathways, one of which is abolished by knockdown of tecpr1a indicating the involvement of sphingomyelin-Tecpr1-induced LC3 lipidation (STIL). Collectively, our observations shed new light on the host immune response against S. pneumoniae , demonstrating that several distinct non-canonical autophagy pathways mediate bacterial degradation by macrophages and providing potential targets for the development of novel therapies to combat pneumococcal infections.