Regulation of the Drosophila Imd pathway by signaling amyloids

Fruit flies elicit effective defense responses against numerous microbes. The responses against Gram-negative bacteria are mediated by the Imd pathway, an evolutionarily conserved NF-kappaB pathway recognizing meso-diaminopimelic acid (DAP)-type peptidoglycan from bacterial cell walls. Several reviews already provide a detailed view of ligand recognition and signal transduction during Imd signaling, but the formation and regulation of the signaling complex immediately downstream of the peptidoglycan-sensing receptors is still elusive. In this review, we focus on the formation of the Imd amyloidal signaling center and post-translational modifications in the assembly and disassembly of the Imd signaling complex

The Imd pathway-mediated immune response in Drosophila

Kohtaamme elinympäristössämme päivittäin monenlaisia mikrobeja. Vaikka useimmat niistä ovat meille haitattomia tai jopa hyödyllisiä, osa voi kuitenkin aiheuttaa sairauksia. Nisäkkäillä, joihin ihminenkin kuuluu, kyky puolustautua mikrobeja vastaan perustuu sekä synnynnäiseen että hankittuun immuniteettiin. Synnynnäinen immuniteetti on välttämätön niin infektioiden ehkäisyssä kuin hankitun immuunivasteen kehityksessä ja säätelyssä. Se perustuu perimän koodaamien proteiinien kykyyn tunnistaa erilaisia mikrobien pintarakenteita ja viestiä tästä soluille signalointireittien välityksellä. Eräs näistä signalointireiteistä, TNFR -signalointireitti, ja sen käynnistämä sytokiinieritys, ovat tarkkaan säädeltyjä ja välttämättömiä normaalille immuunivasteelle. Signaloinnin mekanismeja ja niiden säätelyä ei kuitenkaan vielä täysin tunneta. Synnynnäisen immuniteetin signalointireitit ovat säilyneet hyvin evoluutiossa hyönteisistä ihmisiin. Siksi niiden toimintaa ja säätelyä tutkittaessa voidaan käyttää mallina kodeissakin biojäteastian liepeillä usein tavattavaa banaanikärpästä (Drosophila melanogaster). Banaanikärpäsen Imd-signalointireitti muistuttaa nisäkkäiden TNFR-signalointireittiä. Tässä tutkimusprojektissa pyrimme RNA-häirintää (RNAi) ja muita molekyylibiologian menetelmiä hyödyntäen tunnistamaan Imd-signalointireittiin kuuluvia ja sen säätelyyn osallistuvia proteiineja. Tutkimuksessa löydettiin kolme uutta säätelijää, Tab2, Iap2 ja Pirk. Näiden proteiinien toimintaa ja merkitystä banaanikärpäsen immuunivasteelle selvitettiin tarkemmin. Osoitimme kärpässolumallia apuna käyttäen, että Tab2 ja Iap2 ovat välttämättömiä Imd-signaloinnille. Lisäksi havaitsimme, että banaanikärpäset, joilta Iap2 on poistettu, ovat herkkiä Gram-negatiivisten bakteerien aiheuttamille infektioille. Pirk puolestaan on aiemmin tuntematon proteiini, jonka osoitimme hillitsevän Imd signalointia sekä solumallissa että elävissä kärpäsissä. Pirkin vaikutus oli niin tehokas, että geenin yli-ilmentäminen kärpäsissä riitti herkistämään ne Gram-negatiivisten bakteerien aiheuttamille infektioille. Tehty tutkimus osoittaa, että Imd signalointi on tarkkaan säädeltyä ja aiempaa luultua monimutkaisempaa. Banaanikärpänen mallieläimenä tarjoaa mahdollisuuden tutkia tehokkaasti synnynnäisen immuniteetin säätelyä. Tab2:lla ja Iap2:lla on vastineensa myös nisäkkäissä, joten on mahdollista, että tutkimuksesta saadut tulokset tuovat uusia näkökulmia myös nisäkkäiden immuunisignaloinnin toiminnan selvittämiseen.Innate immunity is the first line of defense against microbes and it is indispensable in preventing infections as well as in the development and regulation of the adaptive immune system. Innate immunity is based on the ability of genome-encoded proteins to recognize and bind microbial surface structures, which is followed by the activation of the innate immune response via various cell signaling pathways. Tumor necrosis factor receptor (TNFR) signaling and cytokine release are strictly regulated and essential for a normal immune response. However, in certain diseases, like infections and autoimmune diseases, cytokines are produced in excess, which prolongs the inflammation and causes tissue damage. Clinical medicine is trying to prevent cytokine overproduction by suppressing TNFR signaling. However, this is challenging, since despite the extensive research carried out in this field in recent years, the mechanisms and regulation of TNFR signaling are not thoroughly understood. The pathways of innate immunity signaling are evolutionarily conserved from insects to humans. Unlike mammals, insects have no adaptive immunity, and hence they are completely dependent on their innate immune response. For these reasons the fruit fly (Drosophila melanogaster) is a suitable model organism for studying innate immunity. In Drosophila, the immune response to Gram-negative bacteria is mediated mainly via the Imd (immune deficiency) signaling pathway, whose intracellular parts resemble the mammalian TNFR signaling pathway. The aim of this research project was to identify and molecularly characterize the components of the Imd pathway and regulatory proteins using a large-scale RNA interference (RNAi) screen. The function and importance of three of the identified regulators, namely Tak1-associated binding protein 2 (Tab2), Inhibitor of apoptosis 2 (Iap2), and Poor Imd response upon knock-in (Pirk), were then further assessed. Using Drosophila S2 cells we showed that Tab2 is essential for both the early and sustained immune responses, while Iap2 mainly regulates the sustained immune response. In addition, we discovered that when Iap2 was removed from fruit flies by in vivo RNAi the flies became susceptible to Gram-negative bacterial infections. Pirk was a previously unknown protein that we demonstrated could suppress the Imd pathway activity both in S2 cells and in flies. The inhibitory action of Pirk was shown to be efficient enough to sensitize the flies to Gram-negative bacterial infections. We found that Pirk interacts with the receptor of the Imd pathway, PGRP-LC (Peptidoglycan recognition protein LC), and the downstream component IMD. However, the elucidation of the exact mechanism of Pirk action requires further studies. The present study demonstrates that Imd signaling is strictly regulated and more complex than was previously thought. Drosophila as a model organism provides tools to efficiently study innate immunity. In addition, the results gained from this research in flies can provide new perspectives and may help understand also the mechanisms of signaling in the mammalian innate immune system

ADAM15 gene structure and differential alternative exon use in human tissues

Background: ADAM15 is a metalloprotease-disintegrin implicated in ectodomain shedding and cell adhesion. Aberrant ADAM15 expression has been associated with human cancer and other disorders. We have previously shown that the alternative splicing of ADAM15 transcripts is mis-regulated in cancer cells. To gain a better understanding of ADAM15 regulation, its genomic organization and regulatory elements as well as the alternative exon use in human tissues were characterized. Results: Human ADAM15, flanked by the FLJ32785/DCST1 and ephrin-A4 genes, spans 11.4 kb from the translation initiation codon to the polyadenylation signal, being the shortest multiple-exon ADAM gene. The gene contains 23 exons varying from 63 to 316 bp and 22 introns from 79 to 1283 bp. The gene appeared to have several transcription start sites and their location suggested the promoter location within a CpG island proximal to the translation start. Reporter expression experiments confirmed the location of functional GC-rich, TATAless and CAATless promoter, with the most critical transcription-supporting elements located - 266 to - 23 bp relative to the translation start. Normal human tissues showed different complex patterns of at least 13 different ADAM15 splice variants arising from the alternative use of the cytosolic-encoding exons 19, 20a/b, and 21a/b. The deduced ADAM15 protein isoforms have different combinations of cytosolic regulatory protein interaction motifs. Conclusion: Characterization of human ADAM15 gene and identification of elements involved in the regulation of transcription and alternative splicing provide important clues for elucidation of physiological and pathological roles of ADAM15. The present results also show that the alternative exon use is a physiological post-transcriptional mechanism regulating ADAM15 expression in human tissues.Peer reviewe

Optimising protein detection with fixable custom oligo-labelled antibodies for single-cell multi-omics approaches

Background and Aim Single-cell RNA sequencing (scRNA-seq) is a powerful method utilising transcriptomic data for detailed characterisation of heterogeneous cell populations. The use of oligonucleotide-labelled antibodies for targeted proteomics addresses the shortcomings of the scRNA-seq-only based approach by improving detection of low expressing targets. However, optimisation of large antibody panels is challenging and depends on the availability of co-functioning oligonucleotide-labelled antibodies. Main Methods and Results We present here a simple adjustable oligonucleotide-antibody conjugation method which enables a desired level of oligo-conjugation per antibody. The mean labelling in the produced antibody batches varied from 1 to 6 oligos per antibody. In the scRNA-seq multimodal experiment, the highest sensitivity was seen with moderate antibody labelling as the high activation and/or labelling was detrimental to antibody performance. The conjugates were also tested for compatibility with the fixation and freeze storage protocols. The oligo-antibody signal was stable in fixed cells indicating the feasibility of a stain, fix, store, and analyse later type of workflow for multimodal scRNA-seq. Conclusions and Implications Optimised oligo-labelling will improve detection of weak protein targets in scRNA-seq multimodal experiments and reduce sequencing costs due to a more balanced amplification of different antibody signals in CITE-seq libraries. Furthermore, the use of a pre-stain, fix, run later protocol will allow for flexibility, facilitate sample pooling, and ease logistics in scRNA-seq multimodal experiments.Peer reviewe

IIV-6 Inhibits NF-kappaB Responses in Drosophila

The host immune response and virus-encoded immune evasion proteins pose constant, mutual selective pressure on each other. Virally encoded immune evasion proteins also indicate which host pathways must be inhibited to allow for viral replication. Here, we show that IIV-6 is capable of inhibiting the two Drosophila NF-kappaB signaling pathways, Imd and Toll. Antimicrobial peptide (AMP) gene induction downstream of either pathway is suppressed when cells infected with IIV-6 are also stimulated with Toll or Imd ligands. We find that cleavage of both Imd and Relish, as well as Relish nuclear translocation, three key points in Imd signal transduction, occur in IIV-6 infected cells, indicating that the mechanism of viral inhibition is farther downstream, at the level of Relish promoter binding or transcriptional activation. Additionally, flies co-infected with both IIV-6 and the Gram-negative bacterium, Erwinia carotovora carotovora, succumb to infection more rapidly than flies singly infected with either the virus or the bacterium. These findings demonstrate how pre-existing infections can have a dramatic and negative effect on secondary infections, and establish a Drosophila model to study confection susceptibility

Functional Characterization of the Infection-Inducible Peptide Edin in Drosophila melanogaster

Drosophila is a well-established model organism for studying innate immunity because of its high resistance against microbial infections and lack of adaptive immunity. In addition, the immune signaling cascades found in Drosophila are evolutionarily conserved. Upon infection, activation of the immune signaling pathways, Toll and Imd, leads to the expression of multiple immune response genes, such as the antimicrobial peptides (AMPs). Previously, we identified an uncharacterized gene edin among the genes, which were strongly induced upon stimulation with Escherichia coli in Drosophila S2 cells. Edin has been associated with resistance against Listeria monocytogenes, but its role in Drosophila immunity remains elusive. In this study, we examined the role of Edin in the immune response of Drosophila both in vitro and in vivo. We report that edin expression is dependent on the Imd-pathway NF-κB transcription factor Relish and that it is expressed upon infection both in vitro and in vivo. Edin encodes a pro-protein, which is further processed in S2 cells. In our experiments, Edin did not bind microbes, nor did it possess antimicrobial activity to tested microbial strains in vitro or in vivo. Furthermore, edin RNAi did not significantly affect the expression of AMPs in vitro or in vivo. However, edin RNAi flies showed modestly impaired resistance to E. faecalis infection. We conclude that Edin has no potent antimicrobial properties but it appears to be important for E. faecalis infection via an uncharacterized mechanism. Further studies are still required to elucidate the exact role of Edin in the Drosophila immune response

Human thymic T cell repertoire is imprinted with strong convergence to shared sequences

A highly diverse repertoire of T cell antigen receptors (TCR) is created in the thymus by recombination of gene segments and the insertion or deletion of nucleotides at the junctions. Using next-generation TCR sequencing we define here the features of recombination and selection in the human TCR alpha and TCR beta locus, and show that a strikingly high proportion of the repertoire is shared by unrelated individuals. The thymic TCRa nucleotide repertoire was more diverse than TCR beta, with 4.1 x 10(6) vs. 0.81 x 10(6) unique clonotypes, and contained nonproductive clonotypes at a higher frequency (69.2% vs. 21.2%). The convergence of distinct nucleotide clonotypes to the same amino acid sequences was higher in TCRa than in TCR beta repertoire (1.45 vs. 1.06 nucleotide sequences per amino acid sequence in thymus). The gene segment usage was biased, and generally all individuals favored the same genes in both TCR alpha and TCR beta loci. Despite the high diversity, a large fraction of the repertoire was found in more than one donor. The shared fraction was bigger in TCR alpha than TCR beta repertoire, and more common in in-frame sequences than in nonproductive sequences. Thus, both biases in rearrangement and thymic selection are likely to contribute to the generation of shared repertoire in humans.Peer reviewe

Characterization of human T cell receptor repertoire data in eight thymus samples and four related blood samples

T cell receptor (TCR) is a heterodimer consisting of TCR alpha and TCR beta chains that are generated by somatic recombination of multiple gene segments. Nascent TCR repertoire undergoes thymic selections where non-functional and potentially autoreactive receptors are removed. During the last years, the development of high-throughput sequencing technology has allowed a large scale assessment of TCR repertoire and multiple analysis tools are now also available. In our recent manuscript, Human thymic T cell repertoire is imprinted with strong convergence to shared sequences [1], we show highly overlapping thymic TCR repertoires in unrelated individuals. In the current Data in Brief article, we provide a more detailed characterization of the basic features of these thymic and related peripheral blood TCR repertoires. The thymus samples were collected from eight infants undergoing corrective cardiac surgery, two of whom were monozygous twins [2]. In parallel with the surgery, a small aliquot of peripheral blood was drawn from four of the donors. Genomic DNA was extracted from mechanically released thymocytes and circulating leukocytes. The sequencing of TCR alpha and TCR beta repertoires was performed at ImmunoSEQ platform (Adaptive Biotechnologies). The obtained repertoire data were analysed applying relevant features from immunoSEQ (R) 3.0 Analyzer (Adaptive Biotechnologies) and a freely available VDJTools software package for programming language R [3]. The current data analysis displays the basic features of the sequenced repertoires including observed TCR diversity, various descriptive TCR diversity measures, and V and J gene usage. In addition, multiple methods to calculate repertoire overlap between two individuals are applied. The raw sequence data provide a large database of reference TCRs in healthy individuals at an early developmental stage. The data can be exploited to improve existing computational models on TCR repertoire behaviour as well as in the generation of new models. (C) 2021 The Authors. Published by Elsevier Inc.Peer reviewe

The Safety and Efficacy of Live Viral Vaccines in Patients With Cartilage-Hair Hypoplasia

Background: Live viral vaccines are generally contraindicated in patients with combined immunodeficiency including cartilage-hair hypoplasia (CHH); however, they may be tolerated in milder syndromes. We evaluated the safety and efficacy of live viral vaccines in patients with CHH. Methods: We analyzed hospital and immunization records of 104 patients with CHH and measured serum antibodies to measles, mumps, rubella, and varicella zoster virus (VZV) in all patients who agreed to blood sampling (n= 50). We conducted a clinical trial (identifier: NCT02383797) of live VZV vaccine on five subjects with CHH who lacked varicella history, had no clinical symptoms of immunodeficiency, and were seronegative for VZV; humoral and cellular immunologic responses were assessed post-immunization. Results: A large proportion of patients have been immunized with live viral vaccines, including measles-mumps-rubella (MMR) (n= 40, 38%) and VZV (n= 10, 10%) vaccines, with no serious adverse events. Of the 50 patients tested for antibodies, previous immunization has been documented with MMR (n= 22), rubella (n= 2) and measles (n= 1) vaccines. Patients with CHH demonstrated seropositivity rates of 96%/75%/91% to measles, mumps and rubella, respectively, measured at a medium of 24 years post-immunization. Clinical trial participants developed humoral and cellular responses to VZV vaccine. One trial participant developed post-immunization rash and knee swelling, both resolved without treatment. Conclusion: No serious adverse events have been recorded after immunization with live viral vaccines in Finnish patients with CHH. Patients generate humoral and cellular immune response to live viral vaccines. Immunization with live vaccines may be considered in selected CHH patients with no or clinically mild immunodeficiency.Peer reviewe

Computational solutions for spatial transcriptomics

Transcriptome level expression data connected to the spatial organization of the cells and molecules would allow a comprehensive understanding of how gene expression is connected to the structure and function in the biological systems. The spatial transcriptomics platforms may soon provide such information. However, the current platforms still lack spatial resolution, capture only a fraction of the transcriptome heterogeneity, or lack the throughput for large scale studies. The strengths and weaknesses in current ST platforms and computational solutions need to be taken into account when planning spatial transcriptomics studies. The basis of the computational ST analysis is the solutions developed for single-cell RNA-sequencing data, with advancements taking into account the spatial connectedness of the transcriptomes. The scRNA-seq tools are modified for spatial transcriptomics or new solutions like deep learning-based joint analysis of expression, spatial, and image data are developed to extract biological information in the spatially resolved transcriptomes. The computational ST analysis can reveal remarkable biological insights into spatial patterns of gene expression, cell signaling, and cell type variations in connection with cell type-specific signaling and organization in complex tissues. This review covers the topics that help choosing the platform and computational solutions for spatial transcriptomics research. We focus on the currently available ST methods and platforms and their strengths and limitations. Of the computational solutions, we provide an overview of the analysis steps and tools used in the ST data analysis. The compatibility with the data types and the tools provided by the current ST analysis frameworks are summarized.</p