Henna Myllymäki

Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway

The Imd signaling cascade, similar to the mammalian TNF‐receptor pathway, controls antimicrobial peptide expression in Drosophila. We performed a large‐scale RNAi screen to identify novel components of the Imd pathway in Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell‐derived cDNA library were analyzed for their effect on Attacin promoter activity in response to Escherichia coli. We identified seven gene products required for the Attacin response in vitro, including two novel Imd pathway components: inhibitor of apoptosis 2 (Iap2) and transforming growth factor‐activated kinase 1 (TAK1)‐binding protein (TAB). Iap2 is required for antimicrobial peptide response also by the fat body in vivo. Both these factors function downstream of Imd. Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in Relish nuclear localization in vitro, suggesting a novel mode of regulation of the Imd pathway. Our results show that an RNAi‐based approach is suitable to identify genes in conserved signaling cascades.

Pirk Is a Negative Regulator of the Drosophila Imd Pathway

NF-κB transcription factors are involved in evolutionarily conserved signaling pathways controlling multiple cellular processes including apoptosis and immune and inflammatory responses. Immune response of the fruit fly Drosophila melanogaster to Gram-negative bacteria is primarily mediated via the Imd (immune deficiency) pathway, which closely resembles the mammalian TNFR signaling pathway. Instead of cytokines, the main outcome of Imd signaling is the production of antimicrobial peptides. The pathway activity is delicately regulated. Although many of the Imd pathway components are known, the mechanisms of negative regulation are more elusive. In this study we report that a previously uncharacterized gene, pirk, is highly induced upon Gram-negative bacterial infection in Drosophila in vitro and in vivo. pirk encodes a cytoplasmic protein that coimmunoprecipitates with Imd and the cytoplasmic tail of peptidoglycan recognition protein LC (PGRP-LC). RNA interference-mediated down-regulation of Pirk caused Imd pathway hyperactivation upon infection with Gram-negative bacteria, while overexpression of pirk reduced the Imd pathway response both in vitro and in vivo. Furthermore, pirk-overexpressing flies were more susceptible to Gram-negative bacterial infection than wild-type flies. We conclude that Pirk is a negative regulator of the Imd pathway.

The Drosophila Imd Signaling Pathway

The fruit fly, Drosophila melanogaster, has helped us to understand how innate immunity is activated. In addition to the Toll receptor and the Toll signaling pathway, the Drosophila immune response is regulated by another evolutionarily conserved signaling cascade, the immune deficiency (Imd) pathway, which activates NF-κB. In fact, the Imd pathway controls the expression of most of the antimicrobial peptides in Drosophila; thus, it is indispensable for normal immunity in flies. In this article, we review the current literature on the Drosophila Imd pathway, with special emphasis on its role in the (patho)physiology of different organs. We discuss the systemic response, as well as local responses, in the epithelial and mucosal surfaces and the nervous system.

Genome-Wide RNA Interference in Drosophila Cells Identifies G Protein-Coupled Receptor Kinase 2 as a Conserved Regulator of NF-κB Signaling

Because NF-κB signaling pathways are highly conserved in evolution, the fruit fly Drosophila melanogaster provides a good model to study these cascades. We carried out an RNA interference (RNAi)-based genome-wide in vitro reporter assay screen in Drosophila for components of NF-κB pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-κB regulators. Of these, nine dsRNA-treatments affect primarily the Toll pathway. G protein-coupled receptor kinase (Gprk)2, CG15737/Toll pathway activation mediating protein, and u-shaped were required for normal Drosomycin response in vivo. Interaction studies revealed that Gprk2 interacts with the Drosophila IκB homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-κB regulation. Morpholino silencing of the zebrafish ortholog of Gprk2 in fish embryos caused impaired cytokine expression after Escherichia coli infection, indicating a conserved role in NF-κB signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-κB reporter activity. Gprk2 RNAi flies are susceptible to infection with Enterococcus faecalis and Gprk2 RNAi rescues Toll10b-induced blood cell activation in Drosophila larvae in vivo. We conclude that Gprk2/GRK5 has an evolutionarily conserved role in regulating NF-κB signaling.

Eye transformer is a negative regulator of Drosophila JAK/STAT signaling

JAK/STAT signaling pathway is evolutionarily conserved and tightly regulated. We carried out a reporter‐based genome‐wide RNAi in vitro screen to identify genes that regulate Drosophila JAK/STAT pathway and found 5 novel regulators. Of these, CG14225 is a negative regulator structurally related to the Drosophila JAK/STAT pathway receptor Domeless, especially in the extracellular domain, and to the mammalian IL‐6 receptor and the signal transducer gp130. CG14225 coimmunoprecipitates with Domeless and its associated kinase hopscotch in S2 cells. CG14225 RNAi caused hyperphosphorylation of the transcription factor Stat92E in S2 cells on stimulation with the Drosophila JAK/STAT pathway ligand unpaired. CG14225 RNAi in vivo hyperactivated JAK/STAT target genes on septic injury and enhanced unpaired‐induced eye overgrowth, and was thus named the eye transformer (ET). In the gastrointestinal infection model, where JAK/STAT signaling is important for stem cell renewal, CG14225/ET RNAi was protective in vivo. In conclusion, we have identified ET as a novel negative regulator of the Drosophila JAK/STAT pathway both in vitro and in vivo, and it functions in regulating Stat92E phosphorylation.—Kallio, J., Myllymäki, H., Grönholm, J., Armstrong, M., Vanha‐aho, L.‐M., Mäkinen, L., Silvennoinen, O., Valanne, S., Rämet, M. Eye transformer is a negative regulator of Drosophila JAK/STAT signaling. FASEBJ. 24, 4467–4479 (2010). www.fasebj.org

Not4 enhances JAK/STAT pathway-dependent gene expression in Drosophila and in human cells

The JAK/STAT pathway is essential for organogenesis, innate immunity, and stress responses in Drosophila melanogaster. The JAK/STAT pathway and its associated regulators have been highly conserved in evolution from flies to humans. We have used a genome‐wide RNAi screen in Drosophila S2 cells to identify regulators of the JAK/STAT pathway, and here we report the characterization of Not4 as a positive regulator of the JAK/STAT pathway. Overexpression of Not4 enhanced Stat92E‐mediated gene responses in vitro and in vivo in Drosophila. Specifically, Not4 increased Stat92E‐mediated reporter gene activation in S2 cells; and in flies, Not4 overexpression resulted in an 8‐fold increase in Turandot M (TotM) and in a 4‐fold increase in Turandot A (TotA) stress gene activation when compared to wild‐type flies. Drosophila Not4 is structurally related to human CNOT4, which was found to regulate interferon‐γ‐ and interleukin‐4‐induced STAT‐mediated gene responses in human HeLa cells. Not4 was found to coimmunoprecipitate with Stat92E but not to affect tyrosine phosphorylation of Stat92E in Drosophila cells. However, Not4 is required for binding of Stat92E to its DNA recognition sequence in the TotM gene promoter. In summary, Not4/CNOT4 is a novel positive regulator of the JAK/STAT pathway in Drosophila and in humans.—Grönholm, J., Kaustio, M., Myllymäki, H., Kallio, J., Saarikettu, J., Kronhamn, J., Valanne, S., Silvennoinen, O., Rämet, M. Not4 enhances JAK/STAT pathway‐dependent gene expression in Drosophila and in human cells. FASEB J. 26, 1239‐1250 (2012). www.fasebj.org

Transcription factor zfh1 downregulates Drosophila Imd pathway.

The fruit fly Drosophila melanogaster has a powerful innate immune system, which culminates on the synthesis of potent antimicrobial peptides (AMPs). This is mainly controlled by two conserved signaling cascades, the Toll and the immune deficiency (Imd) pathways. Like in humans, Drosophila immune responses need to be under tight control at multiple levels to avoid harmful inflammation. We have identified the transcription factor Zn finger homeodomain 1 (zfh1) as a negative regulator of Drosophila Imd signaling. Knocking down zfh1 in Drosophila S2 cells hyperactivates Imd pathway-mediated AMP expression, whereas forced zfh1 expression blocks Imd pathway response downstream of, or parallel to, the Imd pathway transcription factor Relish. In vivo zfh1 RNAi hyperactivates CecropinB induction upon gram-negative bacterial infection. We conclude that zfh1 is an important regulator of the immune response in Drosophila.

The Zebrafish Breathes New Life into the Study of Tuberculosis

Tuberculosis (TB) is a global health emergency. Up to one-third of the world’s population is infected with Mycobacterium tuberculosis, and the pathogen continues to kill 1.5 million people annually. Currently, the means for preventing, diagnosing, and treating TB are unsatisfactory. One of the main reasons for the poor progress in TB research has been a lack of good animal models to study the latency, dormancy, and reactivation of the disease. Although sophisticated in vitro and in silico methods suitable for TB research are constantly being developed, they cannot reproduce the complete vertebrate immune system and its interplay with pathogens and vaccines. However, the zebrafish has recently emerged as a useful alternative to more traditional models, such as mice, rabbits, guinea pigs, and non-human primates, for studying the complex pathophysiology of a mycobacterial infection. The model is based on the similarity between Mycobacterium marinum – a natural fish pathogen – and M. tuberculosis. In both zebrafish larvae and adult fish, an infection with M. marinum leads to the formation of macrophage aggregates and granulomas, which resemble the M. tuberculosis infections in humans. In this review, we will summarize the current status of the zebrafish model in TB research and highlight the advantages of using zebrafish to dissect mycobacterial virulence strategies as well as the host immune responses elicited against them. In addition, we will discuss the possibilities of using the adult zebrafish model for studying latency, dormancy, and reactivation in a mycobacterial infection.

Animal models in tuberculosis research – where is the beef?

Introduction: Tuberculosis (TB) is a major global health problem, and new drugs and vaccines are urgently needed. As clinical trials in humans require tremendous resources, preclinical drug and vaccine development largely relies on valid animal models that recapitulate the pathology of human disease and the immune responses of the host as closely as possible. Areas covered: This review describes the animal models used in TB research, the most widely used being mice, guinea pigs and nonhuman primates. In addition, rabbits and cattle provide models with a disease pathology resembling that of humans. Invertebrate models, including the fruit fly and the Dictyostelium amoeba, have also been used to study mycobacterial infections. Recently, the zebrafish has emerged as a promising model for studying mycobacterial infections. The zebrafish model also facilitates the large-scale screening of drug and vaccine candidates. Expert opinion: Animal models are needed for TB research and provide valuable information on the mechanisms of the disease and on ways of preventing it. However, the data obtained in animal studies need to be carefully interpreted and evaluated before making assumptions concerning humans. With an increasing understanding of disease mechanisms, animal models can be further improved to best serve research goals.

Identification of novel antigen candidates for a tuberculosis vaccine in the adult zebrafish (Danio rerio)

Tuberculosis (TB) remains a major global health challenge and the development of a better vaccine takes center stage in fighting the disease. For this purpose, animal models that are capable of replicating the course of the disease and are suitable for the early-stage screening of vaccine candidates are needed. A Mycobacterium marinum infection in adult zebrafish resembles human TB. Here, we present a pre-clinical screen for a DNA-based tuberculosis vaccine in the adult zebrafish using an M. marinum infection model. We tested 15 antigens representing different types of mycobacterial proteins, including the Resuscitation Promoting factors (Rpf), PE/PPE protein family members, other membrane proteins and metabolic enzymes. The antigens were expressed as GFP fusion proteins, facilitating the validation of their expression in vivo. The efficiency of the antigens was tested against a low-dose intraperitoneal M. marinum infection (≈ 40 colony forming units), which mimics a primary M. tuberculosis infection. While none of the antigens was able to completely prevent a mycobacterial infection, four of them, namely RpfE, PE5_1, PE31 and cdh, led to significantly reduced bacterial burdens at four weeks post infection. Immunization with RpfE also improved the survival of the fish against a high-dose intraperitoneal injection with M. marinum (≈ 10.000 colony forming units), resembling the disseminated form of the disease. This study shows that the M. marinum infection model in adult zebrafish is suitable for the pre-clinical screening of tuberculosis vaccines and presents RpfE as a potential antigen candidate for further studies.