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Preliminary data on a Toll-like receptor from the colonial ascidian Botryllus schlosseri.
No full textToll-like receptors (TLRs) represent a well-known family of conserved pattern recognition receptors the importance of which, in non-self recognition, was demonstrated in both vertebrates and invertebrates. Tunicates represent the vertebrate sister group and, as invertebrates, they rely only on innate immunity for their defense. As regards TLRs, two transcripts have been described and characterized in the solitary species Ciona intestinalis, referred to as CiTLR1 and CiTLR2. Using the Ciona TLR nucleotide sequences, we examined the genome and the available transcriptomes of Botryllus schlosseri looking for similar sequences. We were able to identify a sequence, with similarity to CiTLR2 and, through in silico transduction and subsequent sequence analysis, we studied the domain content of the putative protein. The sequence, called BsTLR, has a TIR and a transmembrane domain, four LLR and two LRR-CT domains. In addition, we analized Bstlr expression in vivo and in vitro, under various experimental conditions and in different phases of the Botryllus blastogenetic cycle. Our data show that, in different phases, there is a change in gene expression and mRNA location, according to the blastogenetic phase
First evidences of a complement system lytic pathway in invertebrates: data from the compound ascidian Botryllus schlosseri.
No full textThe complement system is well studied in mammals, where more than 30 proteins have been described, involved in the activation and regulation of this important humoral effector. However, the evolutionary history of the complement system is not yet fully elucidated and, in recent years, it has been widely demonstrated that complement system is more than just a defender against intruders. For instance, it is important for the clearance of apoptotic cells and corpses. Botryllus schlosseri is a cosmopolitan ascidian, belonging to the phylum Chordata, considered a model organism for the studies of the evolution of the immune system. Studying the complexity of the complement system in Botryllus, we identified a transcript, in our EST collection, coding a protein containing the MACPF (membrane attack complex/perforin) domain, shared by both most of the proteins involved in the lytic pathway and perforins. Invertebrates, we know that perforins are produced by T-lymphocytes and natural killer cells, whereas the activity of the proteins, with the MACPF domain is regulated by the complement component C3/C5.Comparing the domain topology of vertebrateC9 and our protein, called Botryllus C9-like protein (BsC9), a high level of similarity results. To demonstrate that our C9-like protein can be considered a part of the complement system in B. schlosseri we evaluated the expression of BsC9 with respect to C3 activity. Our previous data demonstrates that B. schlosseri C3 is activated by zymosan. We combined the microinjection of zymosan with and without a validated anti-C3 antibody (against human C3) to block the activity of C3. With this approach we studied in, time course, the expression of both BsC3 and BsC9 demonstrating that the anti-C3 antibody is able to inhibit the expression of BsC9. These results are confirmed in both ISH and ICC using the same antibody, and in vitro with the C3 inhibitor compstatin. In addition, a significant (p < 0.05) decrement of labeling with BsC9 antisense riboprobe and validated antibody anti hsC9 is observed when hemocytes are incubated with zymosan and compstatin. Collectively, these results argue in favor of the presence of components of the lytic pathway in our model organism
BsTLR: a new member of the TLR family of recognition proteins from the colonial ascidian Botryllus schlosseri.
Full text linkToll-like receptors (TLRs) represent a well-known family of conserved pattern recognition receptors the importance of which, in non-self recognition, was demonstrated in both vertebrates and invertebrates. Tunicates represent the vertebrate sister group and, as invertebrates, they rely only on innate immunity for their defense. As regards TLRs, two transcripts have been described and characterized in the solitary species Ciona robusta, referred to as CiTLR1 and CiTLR2. Using the Ciona TLR nucleotide sequences, we examined the available transcriptomes of Botryllus schlosseri looking for similar sequences. We were able to identify a sequence, with similarity to CiTLR2 and, through in silico transduction and subsequent sequence analysis, we studied the domain content of the putative protein. The sequence, called BsTLR, has a TIR and a transmembrane domain, four LLR and two LRR-CT domains. In addition, we analised bstlr transcription in vivo and in vitro, under various experimental conditions and in different phases of the Botryllus blastogenetic cycle. Our data show that, in different phases, there is a change in gene transcription and mRNA location, according to the blastogenetic phase
Identification and expression studies of putative stem/progenitor cell markers in the urochordate Botryllus schlosseri
Full text linkIn the colonial ascidian Botryllus schlosseri, a cyclical generation change guarantees the recurrent (weekly at 20°C) renewal of the zooids. During the blastogenetic cycle (i.e., the interval of time between a generation change and the following one), buds progressively grow to the adult size before replacing the old zooids. With the aim of better elucidating the process stem cell differentiation, with particular reference to the genesis of haemocytes during the of the colonial ascidian, we screened the B. schlosseri genome and transcriptome, looking for transcripts/genes showing similarity to vertebrate molecular markers of haematopoietic stem/progenitor cells. On these sequences, after an in silico translation, we performed the phylogenetic reconstruction that, always, returned us the tunicate relevant position, within the protochordates cluster, of vertebrate sister group. The four mammalian orthologous genes, used as markers for the recognition of haematopoietic stem/progenitor cells, identified in B. schlosseri, are bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6. The ISH assay, performed by antisense specific riboprobes, on haemocyte monolayers and colony sections, resulted in a labelling of the sub-endostylar haemolymph lacunae. This results matches previously morphological data that identified the endostyle as a stem cell niche, strengthening our idea to use bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 genes for the identification of haematopoietic stem/progenitor cells in B. schlosseri. Quantitative real time PCR (qRT-PCR) highlighted the over-expression of the considered genes in the mid-cycle phase of the blastogenetic cycle. During this phase, there is the formation of new secondary buds emerging from the primary buds. The higher transcription levels of bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 in the mid-cycle phase reflect the presence of undifferentiated cells involved in proliferative and differentiation events required for the formation of the new blastogenetic generation
Complement-mediated cooperation between immunocytes in the compound ascidian Botryllus schlosseri
Full text linkTwo main kinds of innate immune responses are present in ascidians: phagocytosis and cytotoxicity. They are mediated by two different types of circulating immunocytes: phagocytes and cytotoxic morula cells (MCs). MCs, once activated by non-self-recognition, can stimulate phagocytosis by the release of soluble factors able to act as opsonins. BsC3, the complement C3 homologue, like mammalian C3, contains the thioester bond required to split the molecule into BsC3a and BsC3b. BsC3b likely represents the MC opsonin as it can enhances phagocytosis. The tenet is supported by the observed reduction in phagocytosing cells after exposure of hemocytes to compstatin, a drug preventing C3 activation, or after the bsc3 knockdown by iRNA injection. In addition, the transcript for BsCR1, homologous to mammalian CR1, is present in Botryllus phagocytes and the transcription is modulated during the blastogenetic cycle. MCs also release cytokines (chemokines) able to recruit immunocytes to the infection site. The activity is inhibited by antibodies raised against human TNFa. Since no genes for TNFa are present in the Botryllus genome, the observed activity is probably related to a TNF-domain containing protein, member of the Botryllus complement system. Conversely, activated phagocytes release a rhamnose-binding lectin able to interact with microbial surfaces and act as opsonin. It can also activate MCs by inducing the release of the reported cytokine and stimulate their degranulation. Overall, the results obtained so far indicate the presence of a well-defined cross-talk between the two types of immunocytes during the immune responses of B. schlosseri
Characterization of the complement system in a colonial tunicate: C3 complement receptors and opsonic role of C3
Full text linkThe compound ascidian Botryllus schlosseri is a reliable model organism for the study of immunobiology. As an invertebrate, it relies only on innate immunity for its defense. We already demonstrated the presence, in Botryllus, of homologues of mammalian C3, Bf, MBL and MASP1, referred to as BsC3, BsBf, BsMBL and BsMASP, respectively. All the complement components identified so far, are expressed by morula cells, the most abundant circulating hemocytes. In mammals, once the complement system is activated, a cascade of reactions occurs resulting in the cleavage of the third complement component (C3) to C3a and C3b, the former exerting a chemotactic activity, the latter acting as opsonin and, ultimately, activating the lytic pathway. The best-known receptor for C3a in mammals is C3aR, whereas CR1 is the receptor able to recognize and bind C3b on the microbial surfaces. Here, we describe, in B. schlosseri, new genes showing homology with vertebrate C3aR and CR1, respectively, and studied their transcription in the course of the colonial blastogenetic cycle. In addition, we continued our analysis of the role of C3 in Botryllus immunity by studying the modulation of BsC3 transcription during the colonial blastogenetic cycle and the effect of bsc3 knockdown on immune responses. Results indicate that only morula cells, and no other immunocytes type, are labelled by the antisense probe for BsC3aR, whereas phagocytes and young, undifferentiated cells, known as hemoblasts, are the cells stained by the probe for BsCR1. Both the bsc3ar and bscr1 genes are constitutively transcribed. However, a modulation in the extent of transcription occurs during the colonial blastogenetic cycle as the amount of BsC3aR mRNA abruptly decreased at TO, whereas no differences were observed when EC and MC were compared. This is probably related to the renewing of circulating cells at TO, that are replaced by new, differentiating cells entering the circulation in the same period
Expression study of molecular markers involved in staminality and differentiation in the colonial ascidians Botryllus schlosseri
Full text linkAscidians are invertebrate chordates, members of the subphylum Tunicata that represents the sister group of vertebrates. They offer the opportunity to investigate and compare the behaviour of both embryonic and adult stem cells. Morphological data suggest the presence of undifferentiated haemocytes (haemoblasts) able to proliferate and give rise to terminally differentiated cells. Relevant studies were also carried out in the neural lineage, in which neural progenitor cells regenerate the brain after extirpation. In B. schlosseri, during the cyclical generation change, bud primordial cells, probably deriving from a pool of long-living stem cells, are able to give rise to the neural complex. We screened the B. schlosseri genome and transcriptome, looking for transcripts/genes showing similarity to vertebrate molecular markers of haematopoietic and neural stem cells. Four sequences, orthologous to mammalian transcripts considered markers of haematopoietic progenitor cells, were identified in B. schlosseri. They are: bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6. In situ hybridization on haemocyte monolayers and colony sections, resulted in labelling of cells in the sub-endostylar haemolymph lacunae. This results matches previously morphological data that identified the endostyle as a stem cell niche. Quantitative real time PCR (qRT-PCR) highlighted the over-expression of the considered genes in the mid-cycle phase of the blastogenetic cycle. During this phase, there is the formation of new secondary buds emerging from the primary buds. The high expression levels of bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 genes in the mid-cycle phase reflect the presence of undifferentiated cells involved in proliferative and differentiation events required for giving rise to the new blastogenetic generation. For the neural lineage, we identified and characterised two transcripts orthologues of vertebrate neural stem cell markers (BsSox2 and BsMsi2). We also studied the expression, during the blastogenetic cycle, of a panel of genes already known to be involved in ascidian larvae neurogenesis, i.e., orthologues of Pax2/5/8, Hox1 and Hox3. ISH with riboprobes for BsSox2, BsMsi2, BsPax2/5/8, BsHox1 and BsHox3 revealed a common labelling in the endostyle niche. The presence of bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 transcripts in the cells of the region known to be a stem cell niche, led us to conclude, not only that our probes identified undifferentiated cells but even that in B. schlosseri are probably present a single population of pluripotent stem cells that could differentiate into haematopoietic or neural cells. The qRT-PCR, showed an high expression level in the mid-cycle phase of all the putative neural markers considered. In this phase new secondary buds are produced from primary buds. Each new bud needs its own neural complex and this requires the proliferation of undifferentiated cells to originate neural gland rudiment and cerebral ganglion. Bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 increased their expression associated with these neurogenesis events and this support their involvement in neural stem cell differentiation
Characterisation of the complement system of a colonial tunicate: study of the expression of C3, CR1, C3aR and role of C3 in nonself recognition.
No full textThe complement system is one of the most ancient immune modulation mechanism of bilaterian metazoans. Three complement-activation pathways are known in vertebrates: the classical, the alternative and the lectin pathways; all of them converge on the cleavage of C3. The compound ascidian Botryllus schlosseri is a reliable model organism for the study of immunobiology. It relies only on innate immunity for its defense and immunocytes. Recently, in the same species, we demonstrated of the lectin and alternative pathways. All the complement components identified so far, are expressed by morula cells, the most abundant circulating hemocytes. In mammals, once the complement system is activated, C3 is cleaved to C3a and C3b, the former exerting a chemokine–like activity, the latter acting as opsonin and, ultimately, activating the lytic pathway. The best-known receptor for C3a in mammals is C3aR, whereas CR1 is the receptor able to recognize and bind C3b on the phagocyte surfaces. In the present work, we describe, in B. schlosseri,one genes showing similarity with vertebrate C3aR and three genes with similarity to CR1 (two soluble forms and one transmembrane), and studied their transcription in the course of the colonial blastogenetic cycle. Results indicate that their mRNAs are located in different immunocytes suggesting the presence of a cross-talk between phagocytes and morula cells. In addition, we continued our analysis of the role of C3 in Botryllus immunity by studying the modulation of BsC3 transcription during the colonial blastogenetic cycle and the effect of bsc3 knockdown on immune responses. Only morula cells, and no other immunocytes type, were labelled by the antisense probe for BsC3aR and the soluble CR1s, whereas phagocytes and young, undifferentiated cells known as hemoblasts were the cells stained by the probe for the membrane-linked BsCR1. Both the bsc3ar and bscr1 genes are constitutively transcribed; however, a modulation of transcription occurs during the colonial blastogenetic cycle as the amount of BsC3aR mRNA abruptly decreased at take-over, whereas no differences were observed when early-cycle and mid-cycle were compared. This is probably related to the renewing of circulating cells at TO, when 20-30% of hemocytes undergo cell death by apoptosis and are replaced by new, differentiating cells entering the circulation in the same period
New data on c1qdc from the colonial ascidian botryllus schlosseri
Full text linkIn the compound ascidian Botryllus schlosseri, we recently identified a novel C1q-domain-containing (C1qDC) protein expressed by circulating immunocytes, called BsC1qDC. It has two globular C1q domains and a signal peptide and can act either as an opsonin and facilitate the phagocytosis of nonself particles or as a cytokine and stimulate the degranulation of cytotoxic cells. In the present work, we used a commercial antibody raised against human CTRP4 (hCTRP4) to provide additional evidences of the involvement of this molecule in immune responses. The antibody was validated in immunoblot analysis and recognizes a band corresponding to the expected molecular weight inferred from the analysis of the amino acid sequence of BsC1qDC. The presence of the antibody in the culture medium in phagocytosis and degranulation assays significantly reduced the two responses. In addition, the relationships between complement C3 activation and bsc1qdc transcription was studied using the injection of C3aR agonist in the colonial vasculature
Characterization of the complement system in a colonial tunicate: C3 complement receptors and opsonic role of C3
Full text linkThe complement system is one of the most ancient immune modulator mechanism of bilaterian metazoans. In vertebrates, three complement-activation pathways are known: the classical, the alternative and the lectin pathways: all of them converge on the cleavage of C3. The compound ascidian Botryllus schlosseri is a reliable model organism for the study of immunobiology. As an invertebrate, B. schlosseri relies only on innate immunity for its defense and immunocytes. We already demonstrated the presence, in Botryllus, of homologues of mammalian C3, Bf, MBL and MASP1, referred to as BsC3, BsBf, BsMBL and BsMASP, respectively. All the complement components identified so far, are expressed by morula cells, the most abundant circulatingemocytes. In mammals, once the complement system isactivated, a cascade of reactions that involves proteolysis and polymerization occurs resulting in the cleavage of the third complement component (C3) to C3a and C3b, the former exerting a chemotactic activity, the latter acting as opsonin and, ultimately, activating the lytic pathway. The best-known receptor for C3a in mammals is C3aR, whereas CR1 is the receptor able to recognize and bind C3b on the microbial surfaces. Here, we describe, in B. schlosseri, two newgenes showing homology with vertebrate C3aR and CR1, respectively, and studied their transcription in the course of the colonial blastogeneticcycle. In addition, we continued our analysis ofthe role of C3 in Botryllus immunity by studying the modulation of BsC3 transcription during the colonial blastogenetic cycle and the effect of bsc3 knockdown on immune responses. Results indicate that only morula cells, and no other immunocytes type, are labelled by the antisense probe for BsC3aR, whereas phagocytes and young, undifferentiated cells, known as hemoblasts, are the cells stained by the probe for BsCR1. This suggests the presence of an important cross-talk between these two immunocytes types. Both the bsc3ar and bscr1 genes are constitutively transcribed as almost all morula cells and phagocytes, respectively, resulted labelled by the antisense probe in the ISH assay, independently of their previous challenge with zymosan, a known activator of B. schlosseri hemocytes. However, a modulation in the extent of transcriptionoccurs during the colonial blastogenetic cycle as the amount of BsC3aR mRNA abruptly decreased at TO, whereas no differences were observed when EC and MC were compared. This is probably related to the renewing of circulating cells at TO, when 20-30% of hemocytes undergo cell death by apoptosis and are replaced by new, differentiating cells entering the circulation in the same period
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