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Melanization and Hemocyte Homeostasis  in the Freshwater Crayfish, Pacifastacus leniusculus
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Blood cells or hemocytes play important roles in immunity. They are a major source of many immune-related molecules such as antibodies in adaptive immunity of vertebrates and prophenoloxidase (proPO) in invertebrates. In the crayfish Pacifastacus leniusculus, the proPO-system has been reported to be an important component of immune responses against microorganisms. In this study, several mutant strains of Aeromonas hydrophila were used to reveal that LPS (lipopolysaccharide) is an important factor for the pathogenicity of A. hydrophila, strongly inducing the proPO system and melanization. This proPO activating system is a multistep process, which has to be tightly controlled to avoid the harmful side effects of toxic intermediates. Many regulating factors have been reported to fine-tune the proPO-system. In this study, the cleavage of caspase-1-like activity was shown to be a novel negative regulator of PO activity in crayfish. Moreover, the fragments obtained by cleavage of proPO by the proPO-activating enzyme and caspase-1-like protein increased bacterial clearance. Thus, the peptides generated also have important biological functions.

In addition to being a source of immune proteins, hemocytes also participate in phagocytosis, encapsulation, and nodulation. An infection normally causes a reduction of hemocyte numbers. Consequently, hemocyte homeostasis is important for maintaining appropriate hemocyte numbers in the circulation of the animal. This study shows that the reactive oxygen species level in the anterior proliferation center of crayfish hematopoietic tissue (HPT), together with cell proliferation, was increased during infection. Pl-β-thymosins were proposed to be involved in hemocyte homeostasis by increasing stem cell migration and thus increasing the circulating hemocyte number. Crayfish hemocyte numbers, as well astakine (Ast1 and Ast2) expression in hemocytes and HPT, were previously shown to be under circadian regulation. Here, we show that Ast1, Ast2, and proPO exhibit rhythmic expression in the crayfish brain similarly to their orthologs, prokineticin 1, prokineticin 2 and tyrosinase, respectively, in the zebrafish brain. Tyrosinase expression was detected in zebrafish brain cells while PO-positive cells were identified as hemocytes that had infiltrated into the crayfish brain. Therefore, this information suggests a close relationship between crayfish hemocytes and the crayfish brain as well as vertebrate neurons.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1086
Keyword [en]
melanization, prophenoloxidase, caspase, hematopoiesis, thymosin, astakine, circadian rhythm, reactive oxygen species, tyrosinase, prokineticin
National Category
Immunology
Identifiers
URN: urn:nbn:se:uu:diva-209209ISBN: 978-91-554-8774-4 (print)OAI: oai:DiVA.org:uu-209209DiVA: diva2:656216
Public defence
2013-11-28, Lindahlssalen, EBC, Norbyvägen 18A, Uppsala, 10:00 (English)
Opponent
Supervisors
Available from: 2013-11-06 Created: 2013-10-15 Last updated: 2016-05-09
List of papers
1. Melanization and Pathogenicity in the Insect, Tenebrio molitor, and the Crustacean, Pacifastacus leniusculus, by Aeromonas hydrophila AH-3
Open this publication in new window or tab >>Melanization and Pathogenicity in the Insect, Tenebrio molitor, and the Crustacean, Pacifastacus leniusculus, by Aeromonas hydrophila AH-3
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2010 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 12, e15728- p.Article in journal (Refereed) Published
Abstract [en]

Aeromonas hydrophila is the most common Aeromonas species causing infections in human and other animals such as amphibians, reptiles, fish and crustaceans. Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Several mutant strains of A. hydrophila AH-3 were initially used to study their virulence in two animal species, Pacifastacus leniusculus (crayfish) and Tenebrio molitor larvae (mealworm). The AH-3 strains used in this study have mutations in genes involving the synthesis of flagella, LPS structures, secretion systems, and some other factors, which have been reported to be involved in A. hydrophila pathogenicity. Our study shows that the LPS (O-antigen and external core) is the most determinant A. hydrophila AH-3 virulence factor in both animals. Furthermore, we studied the immune responses of these hosts to infection of virulent or non-virulent strains of A. hydrophila AH-3. The AH-3 wild type (WT) containing the complete LPS core is highly virulent and this bacterium strongly stimulated the prophenoloxidase activating system resulting in melanization in both crayfish and mealworm. In contrast, the ΔwaaE mutant which has LPS without O-antigen and external core was non-virulent and lost ability to stimulate this system and melanization in these two animals. The high phenoloxidase activity found in WT infected crayfish appears to result from a low expression of pacifastin, a prophenoloxidase activating enzyme inhibitor, and this gene expression was not changed in the ΔwaaE mutant infected animal and consequently phenoloxidase activity was not altered as compared to non-infected animals. Therefore we show that the virulence factors of A. hydrophila are the same regardless whether an insect or a crustacean is infected and the O-antigen and external core is essential for activation of the proPO system and as virulence factors for this bacterium.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-141552 (URN)10.1371/journal.pone.0015728 (DOI)000285793200045 ()21206752 (PubMedID)
Available from: 2011-01-12 Created: 2011-01-12 Last updated: 2017-12-11
2. Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity
Open this publication in new window or tab >>Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity
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2014 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 10, no 4, e1004059- p.Article in journal (Refereed) Published
Abstract [en]

Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.

National Category
Immunology
Identifiers
urn:nbn:se:uu:diva-229571 (URN)10.1371/journal.ppat.1004059 (DOI)000342033600027 ()24722332 (PubMedID)
Funder
Swedish Research Council
Available from: 2014-08-11 Created: 2014-08-11 Last updated: 2017-12-05Bibliographically approved
3. Invertebrate hematopoiesis: an anterior proliferation centre as a link between the hematopoietic tissue and the brain
Open this publication in new window or tab >>Invertebrate hematopoiesis: an anterior proliferation centre as a link between the hematopoietic tissue and the brain
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2012 (English)In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 21, no 17, 3173-3186 p.Article in journal (Refereed) Published
Abstract [en]

During evolution, the innate and adaptive immune systems developed to protect organisms from nonself substances. The innate immune system is phylogenetically more ancient and is present in most multicellular organisms, whereas adaptive responses are restricted to vertebrates. Arthropods, lack the blood cells of the lymphoid lineage, and oxygen-carrying erythrocytes, making them suitable model animals to study the regulation of the blood cells of the innate immune system. Many crustaceans have a long life span and need to continuously synthesize blood cells, in contrast to many insects. The hematopoietic tissue (HPT) of Pacifastacus leniusculus provides a simple model to study hematopoiesis because the tissue can be isolated and the proliferation of stem cells and their differentiation can be studied both in vivo and in vitro. Here we demonstrate new findings of a physical link between the HPT and the brain. Actively proliferating cells were localized to an anterior proliferation centre (APC) in the anterior part of the tissue near the area linking the HPT to the brain, whereas more differentiated cells were detected in the posterior part. The central areas of HPT expand in response to lipopolysaccharide-induced blood loss. Cells isolated from the APC divide rapidly and form cell clusters in vitro; conversely, the cells from the remaining HPT form monolayers, and they can be induced to differentiate in vitro. Our findings offer an opportunity to learn more about invertebrate hematopoiesis and its connection to the central nervous system and thereby to obtain new information about the evolution of different blood and nerve cell lineages.

National Category
Immunology
Research subject
Biology
Identifiers
urn:nbn:se:uu:diva-174038 (URN)10.1089/scd.2012.0077 (DOI)000310840500011 ()22564088 (PubMedID)
Funder
Swedish Research Council, 621-2011-4797; 621-2009-5715; 319-2010-6250Formas, 223.2011-606
Available from: 2012-11-06 Created: 2012-05-10 Last updated: 2017-12-07Bibliographically approved
4. beta-Thymosins and Hemocyte Homeostasis in a Crustacean
Open this publication in new window or tab >>beta-Thymosins and Hemocyte Homeostasis in a Crustacean
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 4, e60974- p.Article in journal (Refereed) Published
Abstract [en]

Thymosin proteins are well known for their actin-binding activity. Thymosin beta 4 (T beta 4) has been associated with biological activities in tissue repair and cell migration via interaction with ATP-synthase in vertebrates, while the information of similar thymosin functions in invertebrates is limited. We have shown previously that ATP-synthase is present on the surface of crayfish hematopoietic tissue (HPT) cells, and that astakine 1 (Ast1, an invertebrate cytokine) was found to interact with this beta-subunit of ATP synthase. Here, we identified five different beta-thymosins from Pacifastacus leniusculus, designated Pl-beta-thymosin1-5. The two dominant isoforms in brain, HPT and hemocytes, Pl-beta-thymosin1 and 2, were chosen for functional studies. Both isoforms could bind to the b-subunit of ATP-synthase, and Pl-beta-thymosin1, but not Pl-beta-thymosin2, significantly increased extracellular ATP formation. Moreover, Pl-beta-thymosin1 stimulated HPT cell migration in vitro and Ast1 blocked this effect. Pl-beta-thymosin2 increased the circulating hemocyte number at an early stage after injection. Additionally, in vivo injection of Pl-beta-thymosin1 resulted in significant reduction of reactive oxygen species (ROS) production in crayfish HPT whereas Pl-beta-thymosin2 had a similar but transient effect. Both Pl-beta-thymosins induced the expression of Ast1 and superoxide dismutase (SOD) transcripts, while silencing of endogenous Pl-beta-thymosin 1 and 2 by RNAi resulted in significant reduction of the Ast1 and SOD transcripts. The diverse effects exhibited by Pl-beta-thymosin1 and Pl-beta-thymosin2 indicates that these proteins are involved in a complex interaction that regulates the hematopoietic stem cell proliferation and differentiation.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-200355 (URN)10.1371/journal.pone.0060974 (DOI)000317717300166 ()
Available from: 2013-05-28 Created: 2013-05-27 Last updated: 2017-12-06Bibliographically approved
5. Circadian regulation of melanization and prokineticin homologues is conserved in the brain of freshwater crayfish and zebrafish
Open this publication in new window or tab >>Circadian regulation of melanization and prokineticin homologues is conserved in the brain of freshwater crayfish and zebrafish
2013 (English)In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 40, no 2, 218-226 p.Article in journal (Refereed) Published
Abstract [en]

Circadian clock is important to living organisms to adjust to the external environment. This clock has been extensively studied in mammals, and prokineticin 2 (Prok2) acts as one of the messenger between the central nervous system and peripheral tissues. In this study, expression profiles of Prok1 and Prok2 were investigated in a non-mammalian vertebrate brain, zebrafish, and the expression was compared to the Prok homologues, astakines (Ast1 and Ast2) in crayfish. These transcripts exhibited circadian oscillation in the brain, and Ast1 had similar pattern to Prok2. In addition, the expression of tyrosinase, an enzyme which expression is regulated by E-box elements like in Prok2, was also examined in zebrafish brain and was compared with the expression of prophenoloxidase (proPO), the melanization enzyme, in crayfish brain. Interestingly, the expressions of both Tyr and proPO displayed circadian rhythm in a similar pattern to Prok2 and Ast1, respectively. Therefore, this study shows that circadian oscillation of prokineticin homologues and enzymes involved in melanization are conserved.

National Category
Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-197592 (URN)10.1016/j.dci.2013.03.002 (DOI)000320491100016 ()23500514 (PubMedID)
Funder
Swedish Research Council, 2011-4797
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2013-03-29 Created: 2013-03-29 Last updated: 2017-12-06Bibliographically approved

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