The Role of Galanin during Bacterial Infection in Larval Zebrafish.

Galanin is a peptide that is conserved among different species and plays various roles in an organism, although its entire role is not completely understood. For many years, galanin has been linked mainly with the neurotransmission in the nervous system; however, recent reports underline its role in immunity. Zebrafish (Danio rerio) is an intensively developing animal model to study infectious diseases. In this study, we used larval zebrafish to determine the role of galanin in bacterial infection. We showed that knockout of galanin in zebrafish leads to a higher bacterial burden and mortality during Mycobacterium marinum and Staphylococcus aureus infection, whereas administration of a galanin analogue, NAX 5055, improves the ability of fish to control the infection caused by both pathogens. Moreover, the transcriptomics data revealed that a lower number of genes were regulated in response to mycobacterial infection in gal-/- mutants compared with their gal+/+ wild-type counterparts. We also found that galanin deficiency led to significant changes in immune-related pathways, mostly connected with cytokine and chemokine functions. The results show that galanin acts not only as a neurotransmitter but is also involved in immune response to bacterial infections, demonstrating the complexity of the neuroendocrine system and its possible connection with immunity.

Integrative Analysis of Human Macrophage Inflammatory Response Related to Mycobacterium tuberculosis Virulence.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, kills 1.5 to 1.7 million people every year. Macrophages are Mtb's main host cells and their inflammatory response is an essential component of the host defense against Mtb. However, Mtb is able to circumvent the macrophages' defenses by triggering an inappropriate inflammatory response. The ability of Mtb to hinder phagolysosome maturation and acidification, and to escape the phagosome into the cytosol, is closely linked to its virulence. The modulation of the host inflammatory response relies on Mtb virulence factors, but remains poorly studied. Understanding macrophage interactions with Mtb is crucial to develop strategies to control tuberculosis. The present study aims to determine the inflammatory response transcriptome and miRNome of human macrophages infected with the virulent H37Rv Mtb strain, to identify macrophage genetic networks specifically modulated by Mtb virulence. Using human macrophages infected with two different live strains of mycobacteria (live or heat-inactivated Mtb H37Rv and M. marinum), we quantified and analyzed 184 inflammatory mRNAs and 765 micro(mi)RNAs. Transcripts and miRNAs differently modulated by H37Rv in comparison with the two other conditions were analyzed using in silico approaches. We identified 30 host inflammatory response genes and 37 miRNAs specific for H37Rv virulence, and highlight evidence suggesting that Mtb intracellular-linked virulence depends on the inhibition of IL-1ß-dependent pro-inflammatory response, the repression of apoptosis and the delay of the recruitment and activation of adaptive immune cells. Our findings provide new potential targets for the development of macrophage-based therapeutic strategies against TB.

Glucocorticoid-Induced Exacerbation of Mycobacterial Infection Is Associated With a Reduced Phagocytic Capacity of Macrophages.

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 results show that the synthetic glucocorticoid beclomethasone increases the bacterial burden and the dissemination of a systemic Mm infection. The exacerbated Mm infection was associated with a decreased phagocytic activity of macrophages, higher percentages of extracellular bacteria, and a reduced rate of infected cell death, whereas the bactericidal capacity of the macrophages was not affected. The inhibited phagocytic capacity of macrophages was associated with suppression of the transcription of genes involved in phagocytosis in these cells. The decreased bacterial phagocytosis by macrophages was not specific for Mm, since it was also observed upon infection with Salmonella Typhimurium. In conclusion, our results show that glucocorticoids inhibit the phagocytic activity of macrophages, which may increase the severity of bacterial infections like tuberculosis.

Disruption of Cxcr3 chemotactic signaling alters lysosomal function and renders macrophages more microbicidal.

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 from zebrafish larvae carrying a mutation in a chemokine receptor of the Cxcr3 family display upregulated expression of vesicle trafficking and lysosomal genes and possess enlarged lysosomes that enhance intracellular bacterial clearance. This increased microbicidal capacity is phenocopied by inhibiting the lysosomal transcription factor EC, while its overexpression counteracts the protective effect of chemokine receptor mutation. Tracking macrophage migration in zebrafish revealed that lysosomes of chemokine receptor mutants accumulate in the front half of cells, preventing macrophage polarization during chemotaxis and reaching sites of inflammation. Our work shows that chemotactic signaling affects the bactericidal properties and localization during chemotaxis, key aspects of the inflammatory response.

Hif-1alpha stabilisation is protective against infection in zebrafish comorbid models.

Multi-drug-resistant tuberculosis is a worldwide problem, and there is an urgent need for host-derived therapeutic targets, circumventing emerging drug resistance. We have previously shown that hypoxia-inducible factor-1α (Hif-1α) stabilisation helps the host to clear mycobacterial infection via neutrophil activation. However, Hif-1α stabilisation has also been implicated in chronic inflammatory diseases caused by prolonged neutrophilic inflammation. Comorbid infection and inflammation can be found together in disease settings, and it remains unclear whether Hif-1α stabilisation would be beneficial in a holistic disease setting. Here, we set out to understand the effects of Hif-1α on neutrophil behaviour in a comorbid setting by combining two well-characterised in vivo zebrafish models - TB infection (Mycobacterium marinum infection) and sterile injury (tailfin transection). Using a local Mm infection near to the tailfin wound site caused neutrophil migration between the two sites that was reduced during Hif-1α stabilisation. During systemic Mm infection, wounding leads to increased infection burden, but the protective effect of Hif-1α stabilisation remains. Our data indicate that Hif-1α stabilisation alters neutrophil migration dynamics between comorbid sites and that the protective effect of Hif-1α against Mm is maintained in the presence of inflammation, highlighting its potential as a host-derived target against TB infection.

The Case for Modeling Human Infection in Zebrafish.

Zebrafish (Danio rerio) larvae are widely recognized for studying host-pathogen interactions in vivo because of their optical transparency, genetic manipulability, and translational potential. The development of the zebrafish immune system is well understood, thereby use of larvae enables investigation solely in the context of innate immunity. As a result, infection of zebrafish with natural fish pathogens including Mycobacterium marinum has significantly advanced our understanding of bacterial pathogenesis and vertebrate host defense. However, new work using a variety of human pathogens (bacterial, viral, and fungal) has illuminated the versatility of the zebrafish infection model, revealing unexpected and important concepts underlying infectious disease. We propose that this knowledge can inform studies in higher animal models and help to develop treatments to combat human infection.

High content analysis of granuloma histology and neutrophilic inflammation in adult zebrafish infected with Mycobacterium marinum.

Infection of zebrafish with natural pathogen Mycobacterium marinum is a useful surrogate for studying the human granulomatous inflammatory response to infection by Mycobacterium tuberculosis. The adaptive immune system of the adult stage zebrafish offers an advance on the commonly used embryo infection model as adult zebrafish form granulomas with striking similarities to human-M. tuberculosis granulomas. Here, we present workflows to perform high content analyses of granulomas in adult zebrafish infected with M. marinum by cryosectioning to take advantage of strong endogenous transgenic fluorescence adapted from common zebrafish embryo infection tools. Specific guides to classifying granuloma necrosis and organisation, quantifying bacterial burden and leukocyte infiltration of granulomas, visualizing foam cell formation, analysing extracellular matrix remodelling and granuloma fibrosis are also provided. We use these methods to characterize neutrophil recruitment to M. marinum granulomas across time and find an inverse relation to granuloma necrosis suggesting granuloma necrosis is not a marker of immunopathology in the natural infection system of the adult zebrafish-M. marinum pairing. The methods can be easily translated to studying the zebrafish adaptive immune response to other chronic and granuloma-forming pathogens.

The Mycobacterium marinum ESX-1 system mediates phagosomal permeabilization and type I interferon production via separable mechanisms.

Following mycobacterial entry into macrophages the ESX-1 type VII secretion system promotes phagosomal permeabilization and type I IFN production, key features of tuberculosis pathogenesis. The current model states that the secreted substrate ESAT-6 is required for membrane permeabilization and that a subsequent passive leakage of extracellular bacterial DNA into the host cell cytosol is sensed by the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) pathway to induce type I IFN production. We employed a collection of Mycobacterium marinum ESX-1 transposon mutants in a macrophage infection model and show that permeabilization of the phagosomal membrane does not require ESAT-6 secretion. Moreover, loss of membrane integrity is insufficient to induce type I IFN production. Instead, type I IFN production requires intact ESX-1 function and correlates with release of mitochondrial and nuclear host DNA into the cytosol, indicating that ESX-1 affects host membrane integrity and DNA release via genetically separable mechanisms. These results suggest a revised model for major aspects of ESX-1-mediated host interactions and put focus on elucidating the mechanisms by which ESX-1 permeabilizes host membranes and induces the type I IFN response, questions of importance for our basic understanding of mycobacterial pathogenesis and innate immune sensing.

Infection and RNA-seq analysis of a zebrafish tlr2 mutant shows a broad function of this toll-like receptor in transcriptional and metabolic control and defense to Mycobacterium marinum infection.

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 a model for tuberculosis. We measured infection phenotypes and transcriptome responses using RNA deep sequencing in mutant and control larvae. RESULTS: tlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2+/- control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2-/- larvae than in tlr2+/-, or tlr2+/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2-/- versus tlr2+/- shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b, tnfb, cxcl11aa/ac, fosl1a, and cebpb. Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis. CONCLUSION: The transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2-specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.

Distinct Host-Mycobacterial Pathogen Interactions between Resistant Adult and Tolerant Tadpole Life Stages of Xenopus laevis.

Mycobacterium marinum is a promiscuous pathogen infecting many vertebrates, including humans, whose persistent infections are problematic for aquaculture and public health. Among unsettled aspects of host-pathogen interactions, the respective roles of conventional and innate-like T (iT) cells in host defenses against M. marinum remain unclear. In this study, we developed an infection model system in the amphibian Xenopus laevis to study host responses to M. marinum at two distinct life stages, tadpole and adult. Adult frogs possess efficient conventional T cell-mediated immunity, whereas tadpoles predominantly rely on iT cells. We hypothesized that tadpoles are more susceptible and elicit weaker immune responses to M. marinum than adults. However, our results show that, although anti-M. marinum immune responses between tadpoles and adults are different, tadpoles are as resistant to M. marinum inoculation as adult frogs. M. marinum inoculation triggered a robust proinflammatory CD8+ T cell response in adults, whereas tadpoles elicited only a noninflammatory CD8 negative- and iT cell-mediated response. Furthermore, adult anti-M. marinum responses induced active granuloma formation with abundant T cell infiltration and were associated with significantly reduced M. marinum loads. This is reminiscent of local CD8+ T cell response in lung granulomas of human tuberculosis patients. In contrast, tadpoles rarely exhibited granulomas and tolerated persistent M. marinum accumulation. Gene expression profiling confirmed poor tadpole CD8+ T cell response, contrasting with the marked increase in transcript levels of the anti-M. marinum invariant TCR rearrangement (iVα45-Jα1.14) and of CD4. These data provide novel insights into the critical roles of iT cells in vertebrate antimycobacterial immune response and tolerance to pathogens.

Colony-stimulating factor-1- and interleukin-34-derived macrophages differ in their susceptibility to Mycobacterium marinum.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains the leading global cause of death from an infectious agent. Mycobacteria thrive within their host Mϕs and presently, there is no animal model that permits combined in vitro and in vivo study of mycobacteria-host Mϕ interactions. Mycobacterium marinum (Mm), which causes TB in aquatic vertebrates, has become a promising model for TB research, owing to its close genetic relatedness to Mtb and the availability of alternative, natural host aquatic animal models. Here, we adopted the Xenopus laevis frog-Mm surrogate infection model to study host Mϕ susceptibility and resistance to mycobacteria. Mϕ differentiation is regulated though the CSF-1 receptor (CSF-1R), which is activated by CSF-1 and the unrelated IL-34 cytokines. Using combined in vitro and in vivo approaches, we demonstrated that CSF-1-Mϕs exacerbate Mm infections, are more susceptible to mycobacterial entry and are less effective at killing this pathogen. By contrast, IL-34-Mϕs confer anti-Mm resistance in vivo, are less susceptible to Mm entry and more effectively eliminate internalized mycobacteria. Moreover, we showed that the human CSF-1- and IL-34-Mϕs are likewise, respectively, susceptible and resistant to mycobacteria, and that both frog and human CSF-1-Mϕs are more prone to the spread of mycobacteria and to being infected by Mm-laden Mϕs than the respective IL-34-Mϕ subsets. This work marks the first report describing the roles of these Mϕ subsets in mycobacterial disease and may well lead to the development of more targeted anti-Mtb approaches.

A brief historical and evolutionary perspective on the origin of cellular microbiology research.

Integrated with both a historical perspective and an evolutionary angle, this opinion article presents a brief and personal view of the emergence of cellular microbiology research. From the very first observations of phagocytosis by Goeze in 1777 to the exhaustive analysis of the cellular defence mechanisms performed in modern laboratories, the studies by cell biologists and microbiologists have converged into an integrative research field distinct from, but fully coupled to immunity: cellular microbiology. In addition, this brief article is thought as a humble patchwork of the motivations that have guided the research in my group over a quarter century.

Vaccination of European sea bass Dicentrarchus labrax with avirulent Mycobacterium marinum (iipA::kan mutant).

Mycobacteriosis is a chronic progressive disease affecting teleost fishes all over the world. No vaccine is commercially available against its main etiological agent, Mycobacterium marinum. The mycobacterial gene responsible for invasion and intracellular persistence, iipA, is known to modulate M. marinum pathology. The innate and adaptive immune responses in sea bass (Dicentrarchus labrax) vaccinated with M. marinum iipA::kan mutant with (and without) the use of adjuvant, with (and without) a booster vaccination were monitored. The adjuvanted vaccine induced enhanced immune responses. TNF-α transcription levels were extremely high in spleen of the fish vaccinated with the addition of adjuvant in both fish vaccinated once and twice, followed by an IgM response highly specific for M. marinum. Also, histologically, granulomas started appearing in spleen and head-kidney tissues (but with no visible bacteria) within a month after vaccination, mainly with the adjuvanted vaccine. This was followed by reduction in pathology, as demonstrated by the lower number of granulomas (with visible bacteria), indicating that even heat-killed bacteria were able to elicit granulomatous formations. Adhesion of the internal organs and moderate pigmentation were observed in the perivisceral adipose tissue of nearly all vaccinated fish. Although the adjuvanted heat-killed avirulent iipA::kan mutant clearly induced a strong humoral and adaptive immune response, the booster treatment did not seem to have produced a significantly higher degree of protection from the disease compared to fish that received a single vaccination.

RNAseq Profiling of Leukocyte Populations in Zebrafish Larvae Reveals a cxcl11 Chemokine Gene as a Marker of Macrophage Polarization During Mycobacterial Infection.

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 polarization during inflammation has been visualized by real time imaging in larvae of the zebrafish. This model organism has also become widely used to study macrophage responses to microbial pathogens. To support the increasing use of zebrafish in macrophage biology, we set out to determine the complete transcriptome of zebrafish larval macrophages. We studied the specificity of the macrophage signature compared with other larval immune cells and the macrophage-specific expression changes upon infection. We made use of the well-established mpeg1, mpx, and lck fluorescent reporter lines to sort and sequence the transcriptome of larval macrophages, neutrophils, and lymphoid progenitor cells, respectively. Our results provide a complete dataset of genes expressed in these different immune cell types and highlight their similarities and differences. Major differences between the macrophage and neutrophil signatures were found within the families of proteinases. Furthermore, expression of genes involved in antigen presentation and processing was specifically detected in macrophages, while lymphoid progenitors showed expression of genes involved in macrophage activation. Comparison with datasets of in vitro polarized human macrophages revealed that zebrafish macrophages express a strongly homologous gene set, comprising both M1 and M2 markers. Furthermore, transcriptome analysis of low numbers of macrophages infected by the intracellular pathogen Mycobacterium marinum revealed that infected macrophages change their transcriptomic response by downregulation of M2-associated genes and overexpression of specific M1-associated genes. Among the infection-induced genes, a homolog of the human CXCL11 chemokine gene, cxcl11aa, stood out as the most strongly overexpressed M1 marker. Upregulation of cxcl11aa in Mycobacterium-infected macrophages was found to require the function of Myd88, a critical adaptor molecule in the Toll-like and interleukin 1 receptor pathways that are central to pathogen recognition and activation of the innate immune response. Altogether, our data provide a valuable data mining resource to support infection and inflammation research in the zebrafish model.

Thrombocyte Inhibition Restores Protective Immunity to Mycobacterial Infection in Zebrafish.

BACKGROUND: Infection-induced thrombocytosis is a clinically important complication of tuberculosis infection. Recent studies have highlighted the utility of aspirin as a host-directed therapy modulating the inflammatory response to infection but have not investigated the possibility that the effect of aspirin is related to an antiplatelet mode of action. METHODS: In this study, we utilize the zebrafish-Mycobacterium marinum model to show mycobacteria drive host hemostasis through the formation of granulomas. Treatment of infected zebrafish with aspirin markedly reduced mycobacterial burden. This effect is reproduced by treatment with platelet-specific glycoprotein IIb/IIIa inhibitors demonstrating a detrimental role for infection-induced thrombocyte activation. RESULTS: We find that the reduction in mycobacterial burden is dependent on macrophages and granuloma formation, providing the first in vivo experimental evidence that infection-induced platelet activation compromises protective host immunity to mycobacterial infection. CONCLUSIONS: Our study illuminates platelet activation as an efficacious target of aspirin, a widely available and affordable host-directed therapy candidate for tuberculosis.

Potentiation of P2RX7 as a host-directed strategy for control of mycobacterial infection.

Mycobacterium tuberculosis is the leading worldwide cause of death due to a single infectious agent. Existing anti-tuberculous therapies require long treatments and are complicated by multi-drug-resistant strains. Host-directed therapies have been proposed as an orthogonal approach, but few have moved into clinical trials. Here, we use the zebrafish-Mycobacterium marinum infection model as a whole-animal screening platform to identify FDA-approved, host-directed compounds. We identify multiple compounds that modulate host immunity to limit mycobacterial disease, including the inexpensive, safe, and widely used drug clemastine. We find that clemastine alters macrophage calcium transients through potentiation of the purinergic receptor P2RX7. Host-directed drug activity in zebrafish larvae depends on both P2RX7 and inflammasome signaling. Thus, targeted activation of a P2RX7 axis provides a novel strategy for enhanced control of mycobacterial infections. Using a novel explant model, we find that clemastine is also effective within the complex granulomas that are the hallmark of mycobacterial infection.

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines.

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Humoral and Cellular Immune Response of European Seabass Dicentrarchus labrax Vaccinated with Heat-Killed Mycobacterium marinum (iipA::kan Mutant).

No vaccine is yet commercially available against Mycobacterium marinum, the etiological agent of fish mycobacteriosis (also known as "fish tuberculosis"). The mycobacterial gene responsible for invasion and intracellular persistence, iipA, is known to moderate M. marinum pathology in Zebrafish Danio rerio. Two doses of heat-killed, wild-type, virulent M. marinum and two doses of a heat-killed, avirulent M. marinum iipA::kan mutant strain were used in parallel to vaccinate European Seabass Dicentrarchus labrax. The fish were then challenged with live, virulent M. marinum, and the pathogenesis of the infection was monitored. High specific immunoglobulin M (IgM) response and an increase in cytokine tumor necrosis factor alpha (TNF-α) messenger RNA expression levels were observed in all vaccinated fish. At 1 month postchallenge, TNF-α expression levels increased in spleen tissues of fish vaccinated with the virulent type and in those of unvaccinated fish, whereas in the head kidney, expression was up-regulated only in unvaccinated fish. The expression then decreased, and at 2 months postchallenge, expression appeared similar in all vaccination types. The highest survival rate (75%) was recorded in the group of fish that were vaccinated with a high dose of avirulent iipA::kan mutant. The iipA::kan mutant induced a strong immune response accompanied by only modest tissue disruption. Coupled with an effective program of booster treatments, the iipA::kan mutant vaccine may be developed into a powerful preventive measure against fish mycobacteriosis.