Annemarie H Meijer

• Professor
• Leiden University

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...

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...

Simple Summary This study investigates zebrafish larvae as a model for Mycobacterium tuberculosis infection, a major cause of tuberculosis in humans. Despite not being natural hosts, zebrafish larvae are successfully infected with M. tuberculosis, showing propagation for up to 9 days post-injection using a robotic system for efficiency. Fluorescenc...

The emergence of antimicrobial resistance has created an urgent need for alternative treatments against bacterial pathogens. Here, we investigated kinase inhibitors as potential host-directed therapies (HDTs) against intracellular bacteria, specifically Salmonella Typhimurium (Stm) and Mycobacterium tuberculosis (Mtb). We screened 827 ATP-competiti...

Mycobacterium tuberculosis (Mtb) as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug resistance. The loss of antibiotic efficacy has raised interest in the identification of host-directed therapeutics (HDT) to develop novel treatment strategi...

Zebrafish is a natural host of various Mycobacterium species and a surrogate model organism for tuberculosis research. Mycobacterium marinum is evolutionarily one of the closest non-tuberculous species related to M. tuberculosis and shares the majority of virulence genes. Although zebrafish is not a natural host of the human pathogen, we have previ...

The increasing prevalence of antimicrobial-resistant Staphylococcus aureus strains, especially methicillin-resistant S. aureus (MRSA), poses a threat to successful antibiotic treatment. Unsuccessful attempts to develop a vaccine and rising resistance to last-resort antibiotics urge the need for alternative treatments. Host-directed therapy (HDT) ta...

Anti-bacterial autophagy, also known as xenophagy, is a crucial innate immune process that helps maintain cellular homeostasis by targeting invading microbes. This defense pathway is widely studied in the context of infections with mycobacteria, the causative agents of human tuberculosis and tuberculosis-like disease in animal models. Our previous...

The emergence of antimicrobial resistance has created an urgent need for alternative treatment strategies against deadly bacterial species. In this study, we investigated the potential of kinase inhibitors as host-directed therapies (HDTs) for combating infectious diseases caused by intracellular bacteria, specifically Salmonella Typhimurium (Stm)...

Simple Summary Tuberculosis is a life-threatening disease caused by infection with mycobacteria. These bacteria can grow inside the cells of our immune system, particularly in macrophages, one of the first cells responding to an infection. Macrophages activate several defense mechanisms to combat the infection, but mycobacteria are notorious for es...

The innate immune response to inflammation and infections are complex and represent major challenges for developing much needed new treatments for chronic inflammatory diseases and drug resistant infections. To be ultimately successful, the immune response must be balanced to allow pathogen clearance without excess tissue damage, processes controll...

Damage-Regulated Autophagy Modulator 1 (DRAM1) is an infection-inducible membrane protein, whose function in the immune response is incompletely understood. Based on previous results in a zebrafish infection model, we have proposed that DRAM1 is a host resistance factor against intracellular mycobacterial infection. To gain insight into the cellula...

The global burden of tuberculosis (TB) is aggravated by the continuously increasing emergence of drug resistance, highlighting the need for innovative therapeutic options. The concept of host-directed therapy (HDT) as adjunctive to classical antibacterial therapy with antibiotics represents a novel and promising approach for treating TB. Here, we h...

Damage-Regulated Autophagy Modulator 1 (DRAM1) is an infection-inducible membrane protein, whose function in the immune response is incompletely understood. Based on previous results in a zebrafish infection model, we have proposed that DRAM1 is a host resistance factor against intracellular mycobacterial infection. To gain insight into the cellula...

In Gaucher disease (GD), the deficiency of glucocerebrosidase (GCase/ GBA1) causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase (ACase) to glucosylsphingosine (GlcSph) in the lysosome. Chronically elevated blood and tissue GlcSph is thought to contribute to symptoms in GD patients as well as to i...

The innate immune response to inflammatory stimuli must be finely balanced to produce an appropriate pro-inflammatory response while allowing a subsequent return to homeostasis. In recent years, in vivo transgenic zebrafish models have shed light on the temporal regulation of the pro-inflammatory innate response to immune challenges. However, until...

Cells of the innate immune system continuously patrol the extracellular environment for potential microbial threats that are to be neutralized by phagocytosis and delivery to lysosomes. In addition, phagocytes employ autophagy as an innate immune mechanism against pathogens that succeed to escape the phagolysosomal pathway and invade the cytosol. I...

The zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of granulomas, the inflammatory lesions characteristic of mycobacterial disease. The optically transparent zebrafish larvae provide a window on the initial stages...

The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic dyes are often used for chromosome labelling in vivo. The mode of binding of these dyes to DNA cause its distortion, elongation, and partial unwinding. T...

The pandemic related strains of Vibrio cholerae are known to cause diarrheal disease in animal hosts. These bacteria must overcome rapid changes in their environment, such as the transition from fresh water to the gastrointestinal system of their host. To study the morphological adjustments during environmental transitions, we used zebrafish as a n...

Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter prote...

Glucocorticoids are effective drugs for treating immune-related diseases, but prolonged therapy is associated with an increased risk of various infectious diseases, including tuberculosis. In this study, we have used a larval zebrafish model for tuberculosis, based on Mycobacterium marinum (Mm) infection, to study the effect of glucocorticoids. Our...

Chemotaxis and lysosomal function are closely intertwined processes essential for the inflammatory response and clearance of intracellular bacteria. We used the zebrafish model to examine the link between chemotactic signaling and lysosome physiology in macrophages during mycobacterial infection and wound-induced inflammation in vivo. Macrophages f...

Dram1 is a stress and infection inducible autophagy modulator that functions downstream of transcription factors p53 and NFκB. Using a zebrafish embryo infection model, we have previously shown that Dram1 provides protection against the intracellular pathogen Mycobacterium marinum by promoting the p62-dependent xenophagy of bacteria that have escap...

The chemokine signaling axes CCR2-CCL2 and CXCR3-CXCL11 participate in the inflammatory response by recruiting leukocytes to damaged tissue or sites of infection and are, therefore, potential pharmacological targets to treat inflammatory disorders. Although multiple CCR2 orthosteric and allosteric inhibitors have been developed, none of these compo...

Many bony features of the face develop from endochondral ossification of preexisting collagen-rich cartilage structures. The proper development of these cartilage structures is essential to the morphological formation of the face. The developmental programs governing the formation of the pre-bone facial cartilages are sensitive to chemical compound...

Dysregulation of the inflammatory response in humans can lead to various inflammatory diseases, like asthma and rheumatoid arthritis. The innate branch of the immune system, including macrophage and neutrophil functions, plays a critical role in all inflammatory diseases. This part of the immune system is well-conserved between humans and the zebra...

Endolysosomal vesicle trafficking and autophagy are crucial degradative pathways in maintenance of cellular homeostasis. The transmembrane protein DRAM1 is a potential therapeutic target that primarily localises to endolysosomal vesicles and promotes autophagy and vesicle fusion with lysosomes. However, the molecular mechanisms underlying DRAM1-med...

There is a growing interest in the use of post-fermentation mycelial waste to obtain cell wall chitin as an added-value product. In the pursuit to identify suitable production strains that can be used for post-fermentation cell wall harvesting, we turned to an Aspergillus niger strain in which the kexB gene was deleted. Previous work has shown that...

Modeling human infectious diseases using the early life stages of zebrafish provides unprecedented opportunities for visualizing and studying the interaction between pathogens and phagocytic cells of the innate immune system. Intracellular pathogens use phagocytes or other host cells, like gut epithelial cells, as a replication niche. The intracell...

Glucocorticoids are effective drugs for treating immune-related diseases, but prolonged therapy is associated with an increased risk of various infectious diseases, including tuberculosis. In this study, we have used a larval zebrafish model for tuberculosis, based on Mycobacterium marinum ( Mm ) infection, to study the effect of glucocorticoids. O...

Glucocorticoids are effective anti-inflammatory drugs, but their clinical use is complicated due to the wide range of side effects they induce. Patients requiring glucocorticoid therapy would benefit from more selective glucocorticoid receptor (GR) agonists, capable of attenuating the immune response without causing these side effects. Ginsenosides...

DNA damage regulated autophagy modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 has been implicated in cancer, myocardial infarction, and infectious diseases, but the molecular and cellular functions of this transmembrane protein remain poorly understood. Previously, we have proposed DRAM1 as a host resistance...

Staphylococcus aureus is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against S. aureus, multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutro...

Chitin is an important fungal cell wall component that is cross-linked to β-glucan for structural integrity. Acquisition of chitin to glucan cross-links has previously been shown to be performed by transglycosylation enzymes in Saccharomyces cerevisiae, called Congo Red hypersensitive (Crh) enzymes. Here, we characterized the impact of deleting all...

Phagocytes are highly motile immune cells that ingest and clear microbial invaders, harmful substances, and dying cells. Their function is critically dependent on the expression of chemokine receptors, a class of G-protein-coupled receptors (GPCRs). Chemokine receptors coordinate the recruitment of phagocytes and other immune cells to sites of infe...

The rapid and persistent increase of drug-resistant Mycobacterium tuberculosis (Mtb) infections poses increasing global problems in combatting tuberculosis (TB), prompting for the development of alternative strategies including host-directed therapy (HDT). Since Mtb is an intracellular pathogen with a remarkable ability to manipulate host intracell...

Chemotaxis and lysosomal function are closely intertwined processes essential for the inflammatory response and clearance of intracellular bacteria. We used the zebrafish model to examine the link between chemotactic signaling and lysosome physiology in macrophages during mycobacterial infection and wound-induced inflammation in vivo. Macrophages f...

Macrophages are phagocytic cells from the innate immune system, which forms the first line of host defense against invading pathogens. These highly dynamic immune cells can adopt specific functional phenotypes, with the pro-inflammatory M1 and anti-inflammatory M2 polarization states as the two extremes. Recently, the process of macrophage polariza...

Antimicrobial resistance in tuberculosis (TB) is a public health threat of global dimension, worsened by increasing drug resistance. Host-directed therapy (HDT) is an emerging concept currently explored as an adjunct therapeutic strategy for TB. One potential host target is the ligand-activated transcription factor aryl hydrocarbon receptor (AhR),...

Background: The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb,...

Announcement Cells Special Issue: Autophagy in Antimicrobial Immunity

Background The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as...

Neutrophilic inflammation with prolonged neutrophil survival is common to many inflammatory conditions, including chronic obstructive pulmonary disease (COPD). There are few specific therapies that reverse neutrophilic inflammation, but uncovering mechanisms regulating neutrophil survival is likely to identify novel therapeutic targets. Screening o...

Background The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as...

β-glucosidases (GBA1 [glucocerebrosidase], GBA2, and GBA3) are ubiquitous, essential enzymes. Lysosomal GBA1 and cytosol-facing GBA2 degrade glucosylceramide (GlcCer); GBA1 deficiency causes Gaucher disease (GD), a lysosomal storage disorder characterized by lysosomal accumulation of GlcCer, which is partly converted to glucosylsphingosine (GlcSph)...

Among opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the zebrafish embryo model, we applied a combination of non-invasive real-time imaging and genetic approaches to compare the infectious development of A. fumigatus...

The CXCR3‐CXCL11 chemokine‐signaling axis plays an essential role in infection and inflammation by orchestrating leukocyte trafficking in human and animal models, including zebrafish. Atypical chemokine receptors (ACKRs) play a fundamental regulatory function in signaling networks by shaping chemokine gradients through their ligand scavenging funct...

Background The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as...

Neutrophilic inflammation with prolonged neutrophil survival is common to many inflammatory conditions, including chronic obstructive pulmonary disease (COPD). There are few specific therapies that reverse neutrophilic inflammation, but uncovering mechanisms regulating neutrophil survival is likely to identify novel therapeutic targets. Screening o...

Inflammasomes are cytosolic multiprotein complexes that regulate inflammatory responses to danger stimuli and infection, and their dysregulation is associated with an increasing number of autoinflammatory diseases. In recent years, zebrafish models of human pathologies to study inflammasome function in vivo have started to emerge. Here, we discuss...

Intracellular pathogens such as Salmonella depend on their molecular virulence factors to evade host defense responses like autophagy. Using a zebrafish systemic infection model, we have previously shown that phagocytes, predominantly macrophages, target Salmonella Typhimurium by an autophagy-related pathway known as Lc3-associated phagocytosis (LA...

The CXCR3-CXCL11 chemokine-signaling axis plays an essential role in infection and inflammation by orchestrating leukocyte trafficking in human and animal models, including zebrafish. Atypical chemokine receptors (ACKRs) play a fundamental regulatory function in signaling networks by shaping chemokine gradients through their ligand scavenging funct...

Background The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as...

Glucocorticoid drugs are widely used to treat immune-related diseases, but their use is limited by side effects and by resistance, which especially occurs in macrophage-dominated diseases. In order to improve glucocorticoid therapies, more research is required into the mechanisms of glucocorticoid action. In the present study, we have used a zebraf...

Macrophages are phagocytic cells from the innate immune system, which forms the first line of host defense against invading pathogens. These highly dynamic immune cells can adopt specific functional phenotypes, with the pro-inflammatory M1 and anti-inflammatory M2 polarization states as the two extremes. Recently, the process of macrophage polariza...

Principal Component Analysis of RNA-sequencing samples. (A–C) Principal Component Analysis of 5 and 6 dpf mpeg1:gal4; UAS-Kaede, and mpeg1:mCherry positive and negative samples (A), 5dpf mpx:gfp and lck:gfp positive and negative samples (B), and 20 cell samples of mpeg1:gfp positive cells infected or not with M. marinum GFP (C).

RT-qPCR validation of macrophage and neutrophils markers by RT-qPCR. (A) Expression of chemokine receptors and lysosomal genes in FACS-sorted macrophages (MΦ, mpeg1:mCherry positive) and compared with non-fluorescent cells (neg). All genes tested are present in the zf core dataset. RT-qPCR were performed in two independent biological replicates. Y-...

Gene expression dataset in neutrophils. List of genes enriched (log2FC ≥ 1 and 0 and P-adj < 0.01) in mpx:gfp positive neutrophils compared with fluorescent negative cells. Associated name and description in bold are additional annotations to the Ensembl annotation. For each gene, one relevant term from the Biological Process GO is presented.

Molecular function associated to the zebrafish core macrophage expression dataset. Network visualization of GO analysis enrichment (molecular function category) of genes from the core macrophage expression data set using BiNGO and EnrichmentMap. Node size corresponds to the number of genes associated to the enriched GO term and edge size to the sim...

Correlation between RNA-sequencing samples. (A–C) HeatMap of Pearson correlation coefficient of 5 and 6 dpf mpeg1:gal4; UAS-Kaede, and mpeg1:mCherry positive and negative samples (A), 5dpf mpx:gfp and lck:gfp positive and negative samples (B) and 20 cell samples of mpeg1:gfp positive cells infected or not with M. marinum GFP (C).

Additional gene annotation. List of manually annotated genes added to the Ensembl annotation version 79 from the genome version Zv9.

Statistics on RNAseq samples. Top: TPM table of macrophage, neutrophil, and lymphoid cell population. TPM for each positive and negative samples were computed by using the longest transcript for each gene. Presented results are average between each replicate.

Summary of gene expression and differential expression analysis. Level of gene expression was distributed based on average TPM values among non-expressed (TPM < 3), moderately expressed (3 ≤ TPM < 100) and highly expressed (TPM ≥ 100) for the positive samples and an average of all the negative samples present in this analysis. Fold Change (FC) betw...

Gene expression dataset in lymphoid cells. List of genes enriched (log2FC ≥ 1 and P-adj < 0.01) in lck:gfp positive lymphoid cells compared with fluorescent negative cells. Associated name and description in bold are additional annotations to the Ensembl annotation. For each gene, one relevant term from the Biological Process GO is presented.

List of the human homologs from zebrafish genes. List of the human homologs from the genes enriched in the zebrafish core macrophage expression dataset.

Differential expression analysis between Mycobacterium marinum infected and uninfected zebrafish macrophages. List of genes upregulated (FC > 0 and P-adj < 0.05) and downregulated (FC < 0 and P-adj < 0.05) in mpeg1:mCherry and Mm-GFP double positive macrophages compared with mpeg1:mCherry only positive macrophages. Associated name and description i...

Zebrafish core macrophage expression dataset. The 437 genes enriched (log2FC ≥ 1 and P-adj < 0.01) in at least two of the three mpeg1 positive cell populations compared with their respective negative cell background represent the zebrafish core macrophage expression data set. Associated gene names and descriptions in bold are additional manual anno...

DNA Damage Regulated Autophagy Modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 has been implicated in cancer, myocardial infarction, and infectious diseases, but the molecular and cellular functions of this transmembrane protein remain poorly understood. Previously, we have proposed DRAM1 as a host resistance...

Staphylococcus aureus is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against S. aureus , multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutr...

Mycobacterial pathogens are the causative agents of chronic infectious diseases like tuberculosis and leprosy. Autophagy has recently emerged as an innate mechanism for defense against these intracellular pathogens. In vitro studies have shown that mycobacteria escaping from phagosomes into the cytosol are ubiquitinated and targeted by selective au...

Characterization of optn and p62 mutant lines. (A) Validation of Baf A1 effect on zebrafish by Western blot. Baf A1 treatment at dosages of 20, 100 and 400 nM was performed by incubation for 12h in egg water. The protein samples were extracted from 4 dpf WT larvae (>10 embryos/sample). The blots were probed with antibodies against Lc3 and Actin. (B...

Transient overexpression of full length but not ΔLIR/ΔUBA(N) mRNAs of optn or p62 results in increased recruitment of GFP-Lc3 to Mm clusters. (A) Validation of transient overexpression effect of full length or ΔLIR/ΔUBA(N) deletion mRNAs of optn and p62 by quantitative PCR. mRNAs were injected into the one cell stage of WT embryos and samples were...

Accession numbers of selective autophagy receptors. (DOCX)

Optn and p62 are highly conserved between zebrafish and human. (A) Protein sequence identity of SLRs between zebrafish and human. The percentage identity and similarity was calculated using a Clustal Omega alignment. (B) Alignment of LIR, UBAN and UBA motifs from the Optn and p62 sequences of different vertebrates. Amino acid sequences of the LIR m...

Injection of optn or p62 MO transiently knocks down the corresponding mRNA and protein. (A) Workflow representing the experimental design in (B-E). optn or p62 MOs were injected into one cell stage WT embryos, and injected embryos were collected for confirmation of the knockdown effect by RT-PCR and Western blot analysis (>20 embryos /sample). (B)...

Deficiency of Optn or p62 does not affect the expression of major inflammatory response genes during Mm infection. (A) Inflammatory cytokines/chemokines were detected by quantitative PCR. Total RNA was isolated at 3dpi from optn+/+, optnΔ5n/Δ5n, p62+/+ and p62Δ37n/Δ37n larvae (>10 /sample) from three biological replicates. Mutant larvae were infect...

Zebrafish lines used in this study. (DOCX)

Primers for complementation and amplification of sgRNA. (DOCX)

Primers used in this study. (DOCX)

Target sites for CRISPR/Cas 9 systems. (DOCX)

Optn or p62 mutation reduces autophagosome formation during Mm infection. (A) Representative confocal micrographs of GFP-Lc3 co-localization with Mm clusters in optn+/+, optnΔ5n/Δ5n, p62+/+ and p62Δ37n/Δ37n infected embryos at 1 dpi. The arrowheads indicate the overlap between GFP-Lc3 and Mm clusters. Scale bars, 10 μm. (B) Quantification of the pe...

Developing tumors interact with the surrounding microenvironment. Myeloid cells exert both anti- and pro-tumor functions and chemokines are known to drive immune cell migration towards cancer cells. It is documented that CXCR4 signaling supports tumor metastasis formation in tissues where CXCL12, its cognate ligand, is abundant. On the other hand,...

Macrophages are phagocytic cells from the innate immune system, which forms the first line of host defense against invading pathogens. These highly dynamic immune cells can adopt specific functional phenotypes, with the pro-inflammatory M1 and anti-inflammatory M2 polarization states as the two extremes. Recently, the process of macrophage polariza...

Glucocerebrosidase (GBA) is a lysosomal β‐glucosidase‐degrading glucosylceramide. Its deficiency causes Gaucher disease (GD), a common lysosomal storage disorder. Carrying a genetic abnormality in GBA constitutes at present the largest genetic risk factor for Parkinson's disease (PD). Conduritol B epoxide (CBE), a mechanism‐based irreversible inhib...

Innate immune defense against intracellular pathogens, like Salmonella, relies heavily on the autophagy machinery of the host. This response is studied intensively in epithelial cells, the target of Salmonella during gastrointestinal infections. However, little is known of the role that autophagy plays in macrophages, the predominant carriers of th...