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Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1086 Melanization and Hemocyte Homeostasis in the Freshwater Crayfish, Pacifastacus leniusculus CHADANAT NOONIN ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2013 ISSN 1651-6214 ISBN 978-91-554-8774-4 urn:nbn:se:uu:diva-209209 Dissertation presented at Uppsala University to be publicly examined in Lindahlssalen, EBC, Norbyvägen 18A, Uppsala, Thursday, November 28, 2013 at 10:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Abstract Noonin, C. 2013. Melanization and Hemocyte Homeostasis in the Freshwater Crayfish, Pacifastacus leniusculus. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1086. 48 pp. Uppsala. ISBN 978-91-554-8774-4. 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 proPOactivating 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. Keywords: melanization, prophenoloxidase, caspase, hematopoiesis, thymosin, astakine, circadian rhythm, reactive oxygen species, tyrosinase, prokineticin Chadanat Noonin, Uppsala University, Department of Organismal Biology, Comparative Physiology, Norbyv 18 A, SE-752 36 Uppsala, Sweden. © Chadanat Noonin 2013 ISSN 1651-6214 ISBN 978-91-554-8774-4 urn:nbn:se:uu:diva-209209 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-209209) To my family List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I. Noonin C, Jiravanichpaisal P, Söderhäll I, Merino S, Tomás JM, Söderhäll K. (2010) Melanization and pathogenicity in the insect, Tenebrio molitor, and the crustacean, Pacifastacus leniusculus, by Aeromonas hydrophila AH-3. PLoS ONE 5(12): e15728. II. Jearaphunt M, Noonin C, Jiravanichpaisal P, Nakamura S, Tassanakajon A, Söderhäll I, Söderhäll K. (2013) Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity. (under revision: PloS Pathogens) III. Noonin C, Lin X, Jiravanichpaisal P, Söderhäll K, Söderhäll I. (2012) Invertebrate hematopoiesis: an anterior proliferation center as a link between the hematopoietic tissue and the brain. Stem Cells Dev.. 21(17):3173-3186. IV. Saelee N, Noonin C, Nupan B, Junkunlo K, Phongdara A, Lin X, Söderhäll K, Söderhäll I. (2013) β-thymosins and hemocyte homeostasis in a crustacean. PLoS One, 8(4):e60974. V. Noonin C, Watthanasurorot A, Winberg S, Söderhäll I. (2013) Circadian regulation of melanization and prokineticin homologues is conserved in the brain of freshwater crayfish and zebrafish. Dev. Comp. Immunol. 40(2):218-26. Reprints were made with permission from the respective publishers. Contents Introduction ................................................................................................... 11 Invertebrate immunity .............................................................................. 11 The prophenoloxidase activating system ................................................. 12 Tyrosinase: the vertebrate melanization enzyme ..................................... 14 Caspases and innate immunity ................................................................. 15 Hemocyte and hematopoietic tissue ......................................................... 16 Hematopoiesis .......................................................................................... 18 Circadian regulation of hematopoiesis and immunity. ............................. 21 Objectives ..................................................................................................... 24 Results and Discussion ............................................................................. 24 Melanization and pathogenicity in the insect, Tenebrio molitor, and the crustacean, Pacifastacus leniusculus, by Aeromonas hydrophila AH-3. (Paper I) .................................................................................... 24 Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity (Paper II) .............................................................................................. 25 Invertebrate hematopoiesis: an anterior proliferation center as a link between the hematopoietic tissue and the brain. (Paper III) ................ 27 β-thymosins and hemocyte homeostasis in a crustacean. (Paper IV) .. 29 Circadian regulation of melanization and prokineticin homologues is conserved in the brain of freshwater crayfish and zebrafish. (Paper V).............................................................................................. 31 Concluding remarks ...................................................................................... 33 Svensk sammanfattning ................................................................................ 35 Acknowledgements ....................................................................................... 37 References ..................................................................................................... 39 Abbreviations APC Ast BGBP/βGBP BrdU CARD CZ DD DED Dscam ECM CHF ER eSOD FGF FREP GC GNBP H3 HPT HSC HUVEC IL-1β Imd L-DOPA LG LGBP LPS Lys MBL MIP MZ NF-κB NK cell PAMP PG Anterior proliferation center Astakine β-glucan binding protein 5-bromo-2-deoxyuridine Caspase activation and recruitment domain Cortical zone Death domain Death effector domain Down syndrome cell adhesion molecule. Extracellular matrix Crustacean hematopoietic factor Endoplasmic reticulum Extracellular superoxide dismutase Fibroblast growth factor Fibrinogen-related protein Granular cell Gram-negative bacteria binding protein Histone H3 Hematopoietic tissue Hematopoietic stem cell Human umbilical vein endothelial cell Interleukin-1 beta Immune deficiency L-dihydroxyphenylalanine Lymph gland Lipopolysaccharide- and glucan-binding protein Lipopolysaccharide Lysine Mannose binding lectin Melaniztion inhibition protein Medullary zone nuclear factor kappa-light-chain-enhancer of activated B cells Natural killer cell Pathogen associated molecular pattern Peptidoglycan PGRP Pl PO ppA Prok proPO proPO-casp proPO-ppA PRR PSC PXN RNAi ROS RPE SCN SGC SPE SPH TEP TGase/TG TLR Trol Tβ WT Peptidoglycan recognition receptor Pacifastacus leniusculus Phenoloxidase proPO-activating enzyme Prokineticin Prophenoloxidase Caspase1-cleaved proPO fragments ppA-cleaved proPO fragments Pattern recognition receptor Posterior signaling center Peroxinectin RNA interference Reactive oxygen species Retinal pigment epithelium Suprachiasmatic nuclei Semigranular cell Spätzle-processing enzyme Serine proteinase homologue Thioester-containing protein Transglutaminase Toll-like receptor Terribly reduced optic lobes β thymosin Wild type Introduction The immune system is an essential network that has developed to protect living organisms against the attacks of foreign invaders such as bacteria and parasites. This system has been divided into two major processes: innate and adaptive immunity. Innate immunity is a universal and primordial system that is present in all multicellular organisms, whereas adaptive immunity is found only in vertebrates. The major difference between these two systems is that the innate immune system lacks memory, which is produced in the form of antibodies, which are proteins that are produced by cells of lymphoid lineage within the adaptive immune system (Jiravanichpaisal et al. 2006). The innate system is an important first line of defense that discriminates self from non-self molecules and recognizes conserved molecules produced by invading microorganisms through germline-encoded receptors. This system responds quickly and can be activated immediately upon infection. In vertebrates, a host response stimulated by the innate immune system is required to trigger and guide adaptive immunity for antibody production (Janeway and Medzhitov 2002, Iwazaki and Medzhitov 2010). Underlying proper functioning of both immune systems, blood cells (hemocytes) play a critical role in accomplishing these responses. Many blood cell types have been classified in vertebrates: (i) blood cells that originated from the myeloid lineage and function in oxygen transport, blood clotting and innate immunity, and (ii) those of lymphoid lineage that function in both innate (NK cell) and adaptive immunity. Both myeloid and lymphoid lineages are derived from the same multipotent hematopoietic stem cell. The blood cells of invertebrates are often called hemocytes and are most comparable to vertebrate myeloid cells; invertebrate hemocytes have critical functions in innate immunity (Hartenstein, 2006). Invertebrate immunity Invertebrates are the most abundant group in the animal kingdom. These animals lack the memory and complexity of adaptive immunity and only use innate immunity, which works efficiently to defend against microbial infections. The invertebrate innate immune system is composed of cellular and humoral responses. The cellular responses include phagocytosis and entrap11 ment of small microorganisms by hemocytes, encapsulation of large foreign particles, and cytotoxic reactions. Humoral responses involve antimicrobial peptide production, which is initiated by the activation of the Toll or Imd pathways, releasing of clotting proteins, and activation of enzymatic cascades such as the prophenoloxidase (proPO) systems. These cellular and humoral responses work collaboratively because hemocytes are a major source of molecules required for humoral responses and because humoral molecules also affect hemocyte function (Jiravanichpaisal et al. 2006). Several innate immune processes are triggered by the recognition of invading microorganisms through host pattern recognition receptors (PRRs). These proteins recognize conserved molecular motifs called pathogen associated molecular patterns (PAMPs), which are found in or released from microorganisms (Iwasaki and Medzhitov 2010). Several PRRs have been identified in many invertebrates, such as peptidoglycan recognition proteins (PGRPs), Gram-negative bacteria binding proteins (GNBP), β-glucan binding protein (BGBP or βGBP), lipopolysaccharide- and glucan-binding protein (LGBP), thioester-containing proteins (TEPs), fibrinogen-related proteins (FREPs), masquerade-like proteins/serine proteinase homologues (SPHs), and C-type lectins (Schulenburg et al. 2007, Cerenius et al. 2010a, Hillyer 2010). The binding of PRRs and PAMPs results in activation of downstream processes that lead to cell-mediated destruction of invading microorganisms and/or activation of intracellular signaling cascades and subsequently induce humoral defense reactions (Cerenius et al. 2010a, Hillyer 2010). For example, proteolytic activation of TEPs upon binding to bacteria in mosquitoes has been reported to cause bacterial opsonization, which then triggers phagocytosis (Hillyer 2010). Insect lectins have been shown to stimulate encapsulation, melanization and nodule formation (Koizumi et al. 1999, Yu and Kanost 2004, Ling and Yu 2006). Binding of ficolin-like proteins to Gram-negative bacteria, A. hydrophila and E. coli, enhanced bacterial clearance in crayfish (Wu et al. 2011). Different isoforms of P. leniusculus Dscam have different binding capacity to Gram-positive and Gramnegative bacteria, and the binding promotes nodulation and phagocytosis in the crayfish (Watthanasurorot et al., 2011a). A masquerade-like protein acts as an opsonin in crayfish and shrimp, and SPHs are known to be proPO system stabilizers in insects (Cerenius et al. 2010a). The prophenoloxidase activating system The proPO activating system is an important innate immune response in many invertebrates. Freshwater crayfish became more susceptible to Aeromonas hydrophila infection when proPO was silenced (Liu et al. 2007). In insects, the proPO activating system, together with phagocytosis, has been 12 reported to work as a first defense in eliminating bacteria before antimicrobial peptides are produced by the host (Haine et al. 2008, Schneider and Chambers 2008). The proPO system can be rapidly activated by very small amounts of microbial PAMPs. This activating system is a multistep enzymatic reaction consisting of several proteinases; it culminates in the conversion of phenolic compounds to toxic quinones by the enzyme phenoloxidase and finally to melanin (Cerenius et al. 2008). The proPO system is strictly regulated, and many protein components in this cascade have been identified. Extensive studies of the proPO system have been performed in insects (Manduca sexta and Tenebrio molitor) and crustaceans, especially in the freshwater crayfish (Zhao et al. 2005, Cerenius et al. 2008, Kan et al. 2008, Cerenius et al. 2010b). The proPO system is initiated by the recognition of PAMPs by PRRs. In the insect T. molitor, β-1,3-glucan (a fungal component) can be recognized by GNBP3, and lysine-type peptidoglycan (Lys-PG, a Gram-positive bacteria component) binds to PGRP-SA, which then leads to the formation of a Lys-PG/PGRP-SA/GNBP1 complex. This complex then activates the downstream serine proteinase activation cascade and the final activated protienase called SPE activates the proPO (Kan et al. 2008, Lee et al. 2009, Park et al. 2010). In crustaceans, lipopolysaccharides (LPS, a Gram-negative bacteria component) or β-1,3-glucans (e.g. laminarin) can be recognized by LGPB (Lee et al. 2000). These pathogen component-binding events trigger downstream serine proteinase activation cascades (Figure 1) and stimulate the release of proPO components from hemocytes. The terminal active serine proteinase, which is called the proPO-activating enzyme, (ppA) cleaves proPO to form active PO. Active PO then catalyzes the formation of quinones and melanin. The cytotoxic quinones and reactive intermediates produced during the activation cascade have harmful effects on invading microorganisms (Cerenius et al. 2008, Nappi et al. 2009). In additioin to LPS and β-1,3-glucans, Lys-PG can also activate PO system in the crayfish although no PGRP has been identified and this activation was shown to require LGBP and serine proteinase homologues (Liu et al., 2011). Besides proPO cleavage, the active ppA can also activate properoxinectin (proPXN) into peroxinectin (PXN) that possesses peroxidase, cell adhesion, opsonic, and encapsulation activities (Lin et al., 2007). Then, active PXN binds to invading microorganisms and stimulates hemocyte-microbe binding possibly through an extracellular superoxide dismutase (eSOD) and integrin. This complex is then phagocytosed, and the invading microbes are then destroyed intracellularly (Johansson et al. 1999, Lin et al. 2007, Cerenius et al. 2008,). The proPO system must be tightly regulated to prevent the overproduction of cytotoxic compounds, which in turn can be toxic to the host. Several proteinase inhibitors participate in this regulation. Several serpin serine proteinase inhibitors have been identified to block the activation of the serine proteinase upstream of the cascade in insects (Cerenius et al. 2008, Jiang et 13 al., 2009, Park et al., 2010, Cerenius et al. 2010b, An et al., 2012). In crayfish, the active ppA can be inhibited by a proteinase inhibitor named pacifastin, a protein containing a proteinase inhibitor light chain and a transferrin heavy chain (Hergenhahn et al., 1987, Aspán et al., 1990, Liang et al., 1997). In addition, melanization inhibition proteins (MIPs), which were found in mealworm, crayfish, and shrimp, can block melanization mediated by active PO. These proteins interfere with the formation of melanin from quinone compounds (Zhao et al. 2005, Söderhäll et al. 2009, Anthong et al. 2010). Recently, P. lenuisculus mannose-binding lectin (Pl-MBL) was identified with capacity to bind to bacteria and cause bacteria agglutination. This protein was also shown to inhibit LPS-induced PO activity in a Ca2+-dependent manner (Wu et al., 2013). Moreover, in Paper II of this thesis, caspase-1-like activity was found to act as an additional regulatory mechanism of PO activity. Figure 1. Simplified scheme of the proPO activating system in crustacean. (-) represents inhibition or downregulation. PPR, pattern recognition receptors; PAMP, pathogen associated molecular pattern; ppA, prophenoloxidase-activating enzyme; proPO, prophenonoloxidase; PO, phenoloxidase; PXN, peroxinectin; MIP, melanization inhibition protein. Tyrosinase: the vertebrate melanization enzyme As mentioned above, invertebrate PO catalyzes the melanization reaction and has similar activity to vertebrate tyrosinase, which hydroxylates Ltyrosine to L-DOPA (L-dihydroxyphenylalanine) and oxidizes L-DOPA to 14 dopaquinone during melanogenesis. Arthropod proPO and vertebrate tyrosinase are members of the type III copper protein family (Cerenius et al., 2008, Hearing, 2011, Slominski et al., 2012). However, PO is not closely related to tyrosinase in structure and phylogeny (Cerenius et al., 2008), although both enzymes catalyze the melanization reaction. Tyrosinase is produced in pigment cells, and within the ER-Golgi network, undergoes maturation and sorting into the membrane of an organelle called the melanosome (Wang and Hebert, 2006). In contrast, proPO is produced as a pro-enzyme by hemocytes without a signal peptide for ER; it is released into the hemolymph and activated during an infection (Cerenius et al., 2008, Cerenius et al., 2010b). The mechanism by which proPO is released from hemocytes is still unknown. In vertebrates, the pigment cells that possess tyrosinase are developed from two different origins: melanocytes, which have neural crest origin and the retinal pigment epithelium (RPE), which originates from the optic cup in the developing forebrain (Murisier and Beermann, 2006). A zebrafish study suggested that the presence of tyrosinase in RPE is necessary for bright light adaptation because the loss of tyrosinase function caused a reduction in LDOPA, which is a precursor of the dopamine neurotransmitter needed in the retinal network (Page-McCaw et al., 2004). In humans with oculocutaneous albinism, which is caused by a mutation in genes encoding melaninproducing proteins leading to the lack of melanin in the eyes, have vision impairment (Grønskov et al., 2007). In fish, promoter analysis of tyrosinase gene revealed that it contains five E-box elements, which indicates that tyrosinase expression is under circadian and light regulation (Camp et al., 2003). In addition, it has been reported that the regulation of this gene is conserved from fish to mammals because the promoter of mouse tyrosinase can drive the expression in the medaka fish (Oryzias latipes) and the promoter from fugu (Takifugu rubripes) can drive tyrosinase expression in mice melanocytes and RPE (Murisier and Beermann, 2006). Moreover, Paper V of this thesis demonstrates that circadian regulation of tyrosinase expression in the zebrafish brain and of the invertebrate melanization enzyme (proPO) expression in the crayfish brain is conserved. Caspases and innate immunity Caspases are proteins of the cysteine protease family that cleave with varying specificity behind aspartate residues of substrates. They are produced as inactive pro-enzymes with a prodomain at the N-terminus. Most caspases contain a large subunit and a small subunit. In mammals, several caspases have been identified; each can be classified as apoptotic or inflammatory caspases (Becker et al., 2013, Sollberger et al., 2013). 15 Apoptotic caspases are caspase-3-related; they are subdivided into initiator caspases and effector caspases. The initiator caspases (caspase-2, 8-10) have a long prodomain containing a death domain (DD), which can be either a death effector domain (DED) or a caspase activation and recruitment domain (CARD). These caspases can interact with death signals by forming molecular complexes via the DD and then undergo self-activation. The active initiators subsequently activate downstream effector caspases (caspase3, -6 and -7), which have a non-DD short prodomain. The active effectors then cleave their downstream substrates: over 600 molecules that are affected by caspases have been identified (Martin et al., 2012, Sollberger et al., 2013). The “inflammatory caspases” are placed in the caspase-1 subgroup (caspase-1, -4, -5, and -12); they serve as regulatory molecules of inflammation and are activated by the PRR-containing innate immune complexes known as inflammasomes. Caspase-1 activates downstream substrate proinflammatory cytokines, pro-IL-1β, pro-IL-18, and pro-IL-33 as well as effector caspase-7. Remarkably, for pro-IL-1β, pro-IL-18 and pro-IL-33, which are non-signaling-peptide proteins, caspase-1 does not only activate their function but also facilitates their secretion after activation as well (Martin et al., 2012, Sollberger et al., 2013). Caspase-1 also participates in unconventional secretion of other cytokines which are not its substrates such as IL-1α, fibroblast growth factor-2 (FGF-2), and inflammasome proteins. However, the mechanism by which caspase-1 facilitates these protein secretions is still unknown (Keller et al., 2008, Nikel and Rabouille, 2009, Sollberger et al., 2013). Hemocyte and hematopoietic tissue Hemocytes are key players in invertebrate immunity. As mentioned above, several proteins involved in invertebrate immune responses are produced and secreted by hemocytes. Moreover, hemocytes are the cells that execute cellmediated immune responses such as phagocytosis and encapsulation (Jiravanichpaisal et al. 2006). In mosquitoes, most of the circulating hemocytes (95%) are capable of phagocytosis (Hillyer 2010). Plasmatocytes are the phagocytotic hemocytes in Drosophila; granular cells (GCs) and plasmatocytes are hemocytes capable of phagocytosis in lepidopteran species (Jiang et al. 2010, Jiravanichpaisal et al. 2006). In crustaceans, hyaline cells and semigranular cells (SGCs) are hemocytes capable for phagocytosis, but the SGCs have limited phagocytotic capacity (Johansson et al., 2000, Roulston and Smith, 2011). However, in hemolymph of freshwater crayfish contains very little percentage of hyaline cells, but higher amount of this cell can be found in marine crustaceans (Smith and Söderhäll, 1983a, Smith and 16 Söderhäll, 1983b, Söderhäll and Smith, 1983, Lin and Söderhäll, 2011, Roulston and Smith, 2011). Hemocytes are derived from self-renewing stem cells, termed hematopoietic stem cells (HSC), which are located in a special hematopoietic tissue. These tissues often contain both HSCs and more differentiated progenitor cells (Hartenstein 2006, Meister and Govind 2006). Hematopoietic tissues are located in close association with the coelomic cavity or blood vessels in which they can release hemocytes into the lumen or vessels (Grigorian and Hartenstein, 2013). In crayfish, the hematopoietic tissue (HPT) is located on top of the stomach and has a thin sheet-like structure surrounding the ophthalmic artery lining from the heart passing through the middle of the HPT through the brain (Figure 2). Figure 2. The HPT of crayfish is closely associated with the main dorsal blood vessel. Trypan blue dye was injected into crayfish heart and it was pumped out from the heart through blood vessels. The blue color is the dye in ophthalmic artery. The black lines indicate upper edges of HPT. Two invertebrate animals in which hematopoiesis have been extensively studied are Drosophila melanogaster and the freshwater crayfish Pacifastacus leniusculus. The hematopoietic tissue of Drosophila is a gland-like structure called the lymph gland, which is associated with the dorsal vessel. Progenitor cells are situated close to the dorsal vessel in an area called the medullary zone (MZ); differentiated cells are located in an outer area called the cortical zone (CZ). The posterior signaling center (PSC) serves as a niche that controls the hematopoiesis in the lymph gland (Grigorian and Hartenstein, 2013). The reactive oxygen species (ROS) level is high in MZ (Owusu-Ansah and Banerjee, 2009), and under infection conditions, in PSC (Figure3) (Sinenko et al., 2012). Differentiated cells disperse into the body cavity during an infection and at the onset of metamorphosis; and the lymph gland disappears after metamorphosis (Grigorian et al., 2011). In Paper III of this thesis, crayfish HSCs were shown to be located in the anterior and edge of the HPT, and more differentiated cells moved downward to the posterior and middle of the HPT. High ROS levels were found in the APC under in17 fectious conditions (Figure 3). However, whether the cells migrate out of the HPT through the ophthalmic artery or other areas of crayfish tissue is still unknown. In humans, the hematopoietic tissue is bone marrow. Stem cells are located in an area that has low O2 level at the edge of the bone marrow, closely associated with bone cells (osteoblasts) that serve as an HSC niche. When HSCs are released from the osteoblastic niche, they move against an oxygen gradient and undergo differentiation. When the cells reach the vascular niche in the center of the bone marrow, they become more mature and can migrate into blood vessels or sinusoids (Mohyeldin et al., 2010). . Figure 3. Organization of the hematopoietic organs in Drosophila, crayfish, and human. CZ, cortical zone; MZ, medullary zone; PSC, posterior signaling center; ROS, reactive oxygen species; HSC, hematopoietic stem cell; APC, anterior proliferation center; BM, bone marrow. Brown triangles represent the gradient of HSC. Blue triangle represents the gradient of O2 level. Darker green color represents differentiating cells in human BM that migrate toward the vessel in the middle of the BM. Hematopoiesis Hemocytes have a lifespan from several days to several weeks; therefore, newly produced hemocytes must be released for substitution. The process in which hemocytes are produced is called hematopoiesis, and the rate of this process can be increased on demand, for instance, when increased hemocyte numbers are required for an effective immune response during an infection (Grigorian and Hartenstein, 2013). In a 70-kg human, an estimated 3x105 red blood cells and 3x104 white blood cells are produced per second at a steady state; these rates can be increased several-fold during blood loss or systemic infection (Takizawa et al., 2012). In adult crayfish, new hemocytes are released into the hemolymph at a rate of approximately 10% of the total number of hemocytes each day (Söderhäll et al. 2003). Moreover, the number of circulating hemocytes has 18 been shown to be controlled by circadian regulation. Specifically, the number of hemocytes in crayfish was high in the morning, but lower during the dark period. The number of circulating hemocytes in crayfish was critical for the animal since crayfish were more susceptible to bacterial infections during the night when hemocyte numbers are low (Watthanasurorot et al. 2011b). In addition, in Paper III of this study, it was shown that cell proliferation in HPT increased when the crayfish was injected with LPS. In Drosophila larva, differentiation of lamellocytes, a type of hemocyte involved in encapsulation, and the formation of melanotic capsules can be induced by wasp infestation (Krzemien et al. 2010, Sinenko et al. 2012). Because hemocytes have a key role in invertebrate innate immunity, studies on hemocyte production (hematopoiesis) can improve our understanding of invertebrate immunity. Table 1. Markers for HPT cells and hemocyte types in P. leniusculus. Cell markers Cell types References PCNA CHF proPO RUNX Peroxinectin KPI SOD HPT cells SGC, HPT cells SGC, GC SGC, GC SGC, GC SGC GC Wu et al., 2008 Lin et al., 2011 Smith and Söderhäll, 1983b* Söderhäll, et al., 2003 Liang et al., 1992 Wu et al., 2008 Wu et al., 2008 Pl-MBL GC Wu et al., 2013 *A study in freshwater crayfish Astacus astacus. PCNA, proliferating cell nuclear antigen (proliferation marker); CHF; crustacean hematopoistic factor; proPO, prophenoloxidase; RUNX, Runt family transcription factor; KPI, kazal proteinase inhibitor; SOD, superoxide dismutase; Pl-MBL, Pacifastacus leniusculus mannose-binding lectin; HPT, hematopoietic tissue; SGC, semigranular cell; GC, granular cell. Crayfish hematopoiesis takes place in the HPT located on the dorsal part of the stomach as mentioned above. Based on cell morphology, there are at least 5 cell types representing distinct developmental stages found in crayfish HPT. These cells serve as precursors for two major crayfish hemocyte types: SCGs and GCs (Chaga et al. 1995, Lin and Söderhäll 2011, Wu et al, 2008). These cells are partially differentiated when they are in the HPT and become fully mature hemocytes expressing proPO when they are released from the hematopoietic lobules (Söderhäll et al, 2003). Some protein markers for HPT cells and hemocytes have been reported, as shown in Table 1. Expression of the transcription factor Runx has been reported to be involved in the final differentiation of SGCs and GCs (Lin and Söderhäll 2011, Söderhäll et al. 2003). Similarly, the Drosophila Runx homologue lozenge (lz) is necessary for the development of proPO-expressing crystal cells (Lebestky et al., 2000). 19 Two crayfish hematopoietic cytokines, astakine 1 (Ast1) and astakine 2 (Ast2) have been identified (Söderhäll et al. 2005, Lin et al. 2010). Astakines are found in many crustaceans and in some insects, but not in dipterans such as Drosophila or Anopheles ssp (Lin and Söderhäll 2011). The plasma level of Ast1 in crayfish has been shown to be under circadian control, and this is one reason why the number of circulating hemocytes varies between day and night (Watthanasurorot et al 2011b). Downregulation of Ast1 by RNAi in crayfish caused failure of hemocyte recruitment in circulation after LPS injection (Söderhäll et al. 2005). An injection of recombinant astakine into tiger shrimp (Penaeus monodon) was shown to enhance hematopoietic cell proliferation (Hsiao and Song, 2010). Astakines are homologous to vertebrate prokineticins (Söderhäll et al. 2005, Lin et al. 2010). Mammalian prokineticins include endocrine glandderived vascular endothelial growth factor or prokineticin 1, and amphibian Bv8 homolog or prokineticin 2. They are small secreted peptides with diverse functions such as cell proliferation and differentiation, cell survival, angiogenesis, and hematopoiesis, etc. (Negri et al., 2009). In addition, prokineticin 2 has been reported to have rhythmic expression in suprachiasmatic nuclei (SCN) in mammalian hypothalamus and to act as an output molecule to regulate circadian activities of the peripheral organs (Li et al., 2012). In Paper V of this thesis, prokineticins and astakines were found to have rhythmic expression in zebrafish and crayfish brains, respectively. Ast1 has been reported to cause reduction of membrane transglutaminase (TGase) activity in HPT and thereby possibly plays a role in crayfish HPT by maintaining the HPT cells in their stem cell form in the tissue by regulating HPT cells and extracellular matrix interactions (ECM) (Lin et al. 2008, Lin and Söderhäll, 2011). In mammals, it is known that the extracellular transglutaminase 2 (TG2) has a role in promoting interactions between cells and ECM, which is important for cell spreading, migration and differentiation, etc. Overexpression of TG2 has been reported to increase survival of mesenchymal stem cells after implantation (Belkin, 2011). The crosslink between TG2 and collagen-I is shown to promote human osteoblast and human foreskin dermal fibroblast proliferation and differentiation (Wang and Griffin, 2012). A recent study in Drosophila shows the importance of the extracellular matrix protein heparin sulfate proteoglycan called Terribly Reduced Optic Lobes (Trol) in hematopoiesis; Trol mutant larvae had abnormal lymph gland structure, low proliferation of progenitors and prematuration of hemocytes (Grigorian et al., 2013). Downregulation of Trol could rescue the reduction of progenitor differentiation caused by loss of the function of Drosophila fibroblast growth factor receptor, heartless (Dragojlovic-Munther and Martinez-Agosto, 2013). A study in tiger shrimp (Penaeus monodon) has revealed a role for intracellular transglutaminase together with the antim20 icrobial peptide crustin Pm4 in reducing Ast1 translation in shrimp hemocytes (Chang et al., 2013). Ast1 can also reduce apoptosis in HPT cells by acting through a protein called crustacean hematopoietic factor (CHF) (Lin et al. 2011). Recombinant Ast1 treatment, both in vivo and in vitro, showed stimulation of HPT cell proliferation and differentiation of the SGC lineage, whereas injection of recombinant Ast2 into crayfish resulted in an increase in the GC number, but treatment with this protein in vitro did not induce HPT cell proliferation (Lin et al. 2010). In addition, in Paper IV of this thesis, another protein, Pl-β-thymosin, involved in hematopoiesis and hemocyte homeostasis, has been identified. Vertebrate β-thymosins (Tβ) are known as actin-binding proteins. They participate in cell migration, cell cycle, wound healing and morphogenesis. Tβ4 is the most abundant Tβ found in mammalian cells (Sribenja et al., 2013). It has also been reported to promote differentiation of rat neural progenitor cells and the mouse premature oligodendrocyte cell line, which is indicated by the expression of mature oligodendrocyte markers after Tβ4 treatments (Santra et al., 2012). In addition, Tβ4 can reduce the inflammatory response by decreasing NF-κB activation in stimulated corneal epithelial cells, and it also reduced ROS levels in H2O2-treated fibroblasts by upregulating the expression of antioxidant enzymes, superoxide dismutase and catalase (Gupta et al., 2012). The reduction of ROS levels was also observed after injection of recombinant Pl-β-thymosin 1 (Paper IV). In addition, Pl-β-thymosins can bind to extracellular ATP synthase (Paper IV), which is present on the HPT cell membrane similar to Ast1 (Lin et al.; 2009). It is interesting to note that Tβ4-induced extracellular ATP production has been reported to participate in human cell migration (Freeman et al., 2011). However, the binding of Plβ-thymosins and Ast1 manipulates the ATP production in an opposite way. Therefore, these proteins have been proposed to corporately function in the maintenance of crayfish hematopoiesis. Circadian regulation of hematopoiesis and immunity. The circadian rhythm is regulated by an autonomous, endogenous, and selfsustaining clock with an approximately 24-hour cycle, which is found in almost all organisms. External factors such as temperature change, food intake, or light, etc., have impacts on the circadian rhythm, but environmental light is most effective. This rhythm intimately links living organisms to environmental cues because it is able to synchronize sleep-awake cycle or behaviors with external factors (Wilking et al., 2013). In mammals, it has been 21 suggested that circadian rhythm possibly regulates the expression of approximately 10% of the genes in heart, liver and the SCN; as a consequence, the circadian clock controls many biological activities (Panda et al., 2002, Storch et al., 2002, Scheiermann et al., 2013). Circadian regulation has been extensively studied in mammals: this endogenous clock is known to regulate HSC release from bone marrow. Under steady-state conditions, a number of HSCs are found in the mammalian circulatory system, and this number has been shown to have circadian oscillation. Changes in light exposure time or in the light-dark cycle can affect this rhythm (Méndez-Ferrer, et al., 2008). These effects are directed by the hypothalamus through hormone secretion in which the light signal is transformed into an endocrine signal (Giudice et al., 2010). The center of the molecular clock is located in the SCN of the hypothalamus and regulates the secretion of adrenergic neurotransmitters or hormones from the sympathetic nervous system in bone marrow. These hormones subsequently regulate downstream effects on cell proliferation and the rhythmic level of chemokine CXC ligand 12 (CXCL12), which finally causes the rhythmic release of HSC into blood circulation (Méndez-Ferrer, et al., 2008, Giudice et al., 2010). This circadian trafficking of mammalian HSCs is suggested to be a strategy for animals to maintain blood cell homeostasis throughout their life under normal conditions (Giudice et al., 2010). An impact of the circadian clock on hematopoiesis has also been found in crayfish. Ast1 and Ast2 expression in HPT as well as in the brain exhibited circadian rhythm (Watthanasurorot et al., 2011b, Paper V) similar to the rhythmic expression of their vertebrate homolog prokineticin 2 in SCN (Cheng et al., 2002, Cheng et al., 2005). The rhythmic expression of Ast2 was shown to be regulated by melatonin hormone and the synthesis of melatonin itself is under the control of the light-dark cycle (Watthanasurorot et al., 2013). In addition to the rhythmic level of astakines, the number of circulating hemocytes in crayfish also exhibited circadian oscillation (Watthanasurorot et al., 2011b). As mentioned earlier, hemocytes or blood cells are key effectors of a proper immune response; because hematopoiesis is under circadian regulation, this clock affects the immune system as well. In mammals, it has been reported that the level of cellular (blood cells) and humoral immune components rhythmically changes according to sleep-awake cycle (Scheiermann et al., 2013). For example, in the mouse, the expression of PRR Toll-like receptor 9 (TLR9) in macrophages was shown to be under the control of a molecular clock (Silver et al., 2012). Moreover, although there is no report of circadian regulation of hematopoiesis in fruit flies, the susceptibility of Drosophila to infection has been shown to be influenced by circadian rhythm (Shirasu-Hiza et al., 2007, Lee and Edery, 2008). In addition, phagocytosis was found to be diminished in flies lacking a core clock protein, timeless (Stone et al., 2012). The circadian clock has also been shown to have impact on the vulnerability of crayfish to infection; this impact is related to cir22 cadian hematopoiesis as indicated by the rhythmic level of astakines and hemocyte numbers (Watthanasurorot et al., 2011b). Furthermore, in Paper V of this thesis, it is shown that proPO, a marker for mature hemocytes and an important immune protein, exhibited rhythmic expression in the crayfish brain. 23 Objectives The melanization and prophenoloxidase-activating system are effective innate immune responses in invertebrates; however, overstimulation of this system can cause harmful effects to the host itself. Therefore, the purpose of this study was to gain a greater understanding of the tight regulation of this immune response. The hemocyte is known to be a source of the proPO system components and plays important roles in immunity, both in cellular and humoral immune responses. An appropriate number of hemocytes must be maintained under normal conditions, and the production of hemocytes needs to be able to be increased on demand during circumstances such as an infection. Thus, this study also aimed to gain more knowledge of hematopoiesis, the production of hemocytes, and hemocyte homeostasis in the crayfish, Pacifastacus leniusculus. Results and Discussion Melanization and pathogenicity in the insect, Tenebrio molitor, and the crustacean, Pacifastacus leniusculus, by Aeromonas hydrophila AH-3. (Paper I) Previously, PO activity was found to be essential for crayfish immune defense against A. hydrophila infection (Jiravanichpaisal et al. 2009, Liu et al. 2007). This bacterium is an abundant Gram-negative bacterium in aquatic environments and a pathogen to both terrestrial and aquatic animals. It is one of the most common Aeromonas species that cause human diseases such as gastroenteritis, wound infection (commonly when injury happens in a freshwater environment), and septicemia in immunocompromised patients (Parker and Shaw, 2011). In addition, infection with this bacterium is a major problem in carp aquaculture in India (Sahoo et al. 2008). Furthermore, A. hydrophila was isolated from cultured rainbow trout and found to be resistant to antibiotics used in aquaculture in Australia (Akinbowale et al. 2007). Recently, it was also isolated from the freshwater crayfish (P. leniusculus) and was found to be highly virulent to this animal (Jiravanichpaisal et al. 2009). Several mutant strains of A. hydrophila AH-3 were used to study their virulence in two different animal species, P. leniusculus and the larvae of T. molitor (mealworm). The A. hydrophila AH-3 strains used in this study have 24 mutations in genes involved in the synthesis of flagella, LPS structures, secretion systems, and certain other factors, which have been reported to be involved in A. hydrophila pathogenicity (Rabaan et al. 2001, Sha et al. 2005, Seshadri et al. 2006, Sha et al. 2007, Sierra et al. 2007, Suarez et al. 2008). Among all mutant strains used, the AH-3 ∆waaE mutant (O:34 antigen and external core negative strain) (Jiménez et al. 2008) was the only strain in which virulence was completely abrogated compared to the AH-3 WT (wildtype). This result, therefore, indicates that O-antigen and the external core of the LPS molecule are the most important factors for the virulence of A. hydrophila AH-3 in our animal models. LPS has previously been reported to be involved in resistance of A. hydrophila to host defense systems by reducing the binding capacity of host complement components to this bacterium (Merino et al. 1991, Nogueras et al. 2000). The clearance that resulted in this study correlates well with the previous reports by demonstrating that the ∆waaE mutant was completely cleared from the mealworm body within 12 h after injection, whereas WT bacteria rapidly propagated further. WT A. hydrophila strongly induced the proPO-activating cascade because melanization was easily observed in the mealworm, and PO activity was significantly increased in crayfish after the infection. In contrast, melanization could not be observed in the ∆waaE mutant-infected mealworms, and the PO activity was slightly increased in crayfish injected with ∆waaE. The expression level of pacifastin can explain the change in the level of PO activity because pacifastin is an inhibitor of ppA (Liang et al. 1997). Pacifastin mRNA levels were down regulated in the WT infected crayfish, but no change could be observed in those infected with ∆waaE mutants. This result confirms the previous study showing that RNAi of pacifastin caused induction of PO activity in crayfish (Liu et al. 2007). In this study, we clearly show that O-antigen and the external core of LPS are the most important factors for the virulence of A. hydrophila AH-3 in our animal models. A. hydrophila AH-3 WT, containing a complete LPS core, strongly stimulated the proPO-activating system and melanization in P. leniusculus and T. molitor larvae, respectively. Because WT AH-3 propagated very rapidly in the host, but mutant AH-3, which lacks LPS core and O-antigen, was eliminated from the host very quickly, we conclude that he LPS core and O-antigen most likely protected the bacteria from other cellular immune responses. Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity (Paper II) Mammalian caspase-1 has been reported to be involved in innate immune responses: its activation is promoted by innate immune complexes called 25 inflammasomes and its activity is required for proteolytic activation and secretion of inflammatory cytokines such as pro-interleukin-1β (IL-1β), IL1α and pro-IL-18 (Keller et al., 2008, Vladimer et al., 2013). In this study, by using an antibody raised against shrimp caspase, the caspase was detected in crayfish hemocyte lysate and the protein level of this caspase was affected by bacterial infection. In addition, two potential cleavage sites of caspase-1 were found in the proPO sequence after Asp363 and Asp389, which would give rise to 2 Nterminal fragments with the predicted sizes of 42 kDa (proPO-casp1) and 45 kDa (proPO-casp2), respectively (Figure 4). These two caspase-1 cleavage sites are downstream of the ppA cleavage site (Arg176), which gives rise to an approximately 20-kDa N-terminal fragment (proPO-ppA) and an approximately 60-kDa C-terminal active PO (Aspán et al., 1995). Therefore, cleavage by caspase-1-like activity has the potential to reduce PO activity, and caspase-1-like activity is hypothesized to serve as a negative regulator of the proPO-activating system. Figure 4. Amino acid sequence of proPO with cleavage sites of ppA (underline) and putative cleavage sites for a caspase-1-like protein (in boxes). Both proPO-casp1 and proPO-casp2 fragments could be detected in the crayfish plasma at 1 and 3 hours after a bacterial injection. Interestingly, the levels of the fragments at 1 h after the virulent A. hydrophila injection were lower than that of non-virulent E. coli injection. In agreement with the fragment levels, the plasma PO activity in the A. hydrophila-injected crayfish significantly increased at 1 h and decreased at 3 h, but there was no significant change in plasma PO activity in E. coli injected crayfish. In addition, the in vitro study showed that the release of the proPO-casp fragments is Ca2+-dependent and can be inhibited by the caspase-1 inhibitor, Z-YVADFMK. These data support the hypothesis that caspase-1-like activity acts as a negative regulator of PO activity. 26 Although the cleavage of caspase-1-like protein causes a reduction of PO activity, crayfish immunity is not compromised because the obtained fragments can still function in the crayfish immune response while the cleavage merely acts to block “overstimulation” of the melanization reaction. Our in vivo study showed that the proPO-casp fragments enhanced bacterial clearance when the recombinant proteins, together with E. coli, were injected into the crayfish. Moreover, the small N-terminal fragment generated by the cleavage of ppA (proPO-ppA) also increased the E. coli clearance. However, the in vitro study revealed that only the proPO-ppA exhibits antibacterial activity. The proPO-ppA caused bacterial agglutination, bacterial death, and finally, a reduction of bacterial number when E. coli was incubated with the recombinant protein in vitro. This finding is similar to the case of human extracellular superoxide dismutase (SOD) in which the C-terminal peptide possesses antimicrobial activity (Pasupuleti et al., 2009). The effect of proPO-casp fragments on reducing bacterial numbers could only be observed in vivo but not in vitro. These results suggest that the peptides themselves cannot directly kill the bacteria, and they most likely stimulate the immune response by recruiting other proteins in the crayfish to facilitate bacterial clearance. Invertebrate hematopoiesis: an anterior proliferation center as a link between the hematopoietic tissue and the brain. (Paper III) Crayfish hemocytes are produced throughout the animal’s lifetime in the hematopoietic tissue (HPT). This tissue was previously observed as a thin sheet-like structure located at the dorsal part of the stomach only (Chaga et al. 1995, Lin et al. 2011). In this study, the crayfish HPT was found to extend towards the anterior part of the crayfish, connecting with the brain (Figure 5). A later study in the Procambarus clarkia crayfish also shows that the most anterior part of the HPT is situated in auxiliary heart, an area close to the brain that supplies blood to the brain and eyes (Chaves da Silva et al., 2013). The morphological link between these two organs possibly supports crosstalk between the nervous system and the hematopoietic tissue. The number of circulating hemocytes and the plasma level of Ast1 in crayfish have previously been shown to be under circadian control (Watthanasurorot et al. 2011b). This indicates that there is an interaction between the crayfish brain and HPT. Moreover, HPT cells have recently been suggested to serve as a source for neuronal precursor cells (Beltz et al. 2011, Benton et al. 2011). This crosstalk has also been reported in mammals (Giudice et al. 2010, Smaaland et al. 2002, Spiegel et al. 2008). In Drosophila larvae, the peripheral nervous system (PNS) has been reported to be residence for circu27 lating hemocytes and support the hemocytes survival (Makhijani et al. 2011). A difference in morphology and proliferation of cells in the anterior and dorsal/posterior part of the HPT was found in this study. The most anterior part of the HPT connecting to the brain is mainly composed of cells with loose chromatin structure (euchromatin). In contrast, dorsal HPT consists of a high percentage of cells with highly condensed chromatin (heterochromatin). The chromatin structure is usually less condensed in embryonic stem cells, becoming more condensed when the cells undergo differentiation (Meshorer and Misteli 2006). Consequently, isolated cells from the most anterior part actively divided and formed cell clusters in vitro, whereas the cells for the rest of the HPT formed monolayers and proliferated more slowly. BrdU incorporation and detection of phosphorylated histone H3 (mitotic cell marker) also revealed that the cells in the anterior part of HPT were more actively proliferating than the cells in the dorsal parts of the tissue. Figure 5. Physical connection of crayfish HPT and brain. APC, anterior proliferation center; HPT, hematopoietic tissue. The proPO transcript is highly expressed in mature hemocytes, but a very low percentage of HPT cells express this transcript (Söderhäll et al. 2003). In the present study, PO-positive cells were mainly found as mature hemocytes present in the ophthalmic artery and in a small population of free HPT cells in the posterior part of the HPT. In Drosophila, proPO expression has been found only in the differentiated cells called crystal cells; these cells reside in the cortical zone of the lymph gland, which is the residence of Drosophila differentiating cells (Krzemien et al. 2010). From these results, we propose that the undifferentiated crayfish HSCs reside in the most anterior part of the HPT and the differentiated cells reside in the dorsal/posterior HPT. Therefore, the most anterior part of HPT was named “Anterior Proliferation Center (APC)” (Figure 5). Further study in Procambarus clarkia provides the information that confirms the stem cell characters of the cells in APC (Chaves da Silva et al., 2013). 28 LPS or laminarin injection mimics what happens during an infection: at early stages, hemocytes are lost from circulation, and these lost hemocytes have to be replaced by newly synthesized hemocytes. In this study, proliferation of the cells in anterior HPT was increased 30 min after LPS/laminarin injection. This result correlates well with a previous study showing higher hemocyte production and release of hemocytes from the HPT after LPS or laminarin injection (Söderhäll et al. 2003, Söderhäll et al. 2005). A similar response has been reported in mice, in which, after bleeding or LPS injection, the release of hematopoietic progenitors from the bone marrow into the blood circulation also increased (Kollet et al. 2006, Spiegel et al. 2008). This situation has also been reported in Drosophila, in which the mitotic index in the lymph gland increased after wasp infestation (Krzemien et al. 2010). Reactive oxygen species (ROS), byproducts of aerobic metabolism, have been shown to be involved in hematopoietic reconstitution or repopulation after transplantation in mice (Lewandowski et al. 2010). These molecules have also been reported to affect the maintenance of HSC quiescence in mice (Ito et al. 2006). In the Drosophila lymph gland, ROS production was found at high levels in the medullary zone and was proposed to prime the hemocyte progenitor cells to differentiate into all hemocyte types (OwusuAnsah and Banerjee 2009). Moreover, an elevation of ROS levels in the posterior signaling center (HSC niche) of the Drosophila lymph gland was recently found to be important for lamellocyte differentiation during wasp infestation (Sinenko et al. 2012). In our study, high ROS levels were detected in the crayfish APC, and the same phenomenon was later found in another crayfish species, Procambarus clarkia (Chaves da Silva et al., 2013). The ROS levels in the APC were found to be elevated when the amount of circulating hemocytes was decreased by either LPS/laminarin injection or as a circadian response. This high ROS level is possibly a result of a high metabolic rate of the rapidly proliferating cells in that area, and this could serve as a unique microenvironment or niche to maintain the HSC behavior in crayfish. However, it cannot be concluded whether the increase in ROS levels is essential for proliferation and differentiation or if this is only a result of a high metabolic rate during the proliferation and differentiation process. β-thymosins and hemocyte homeostasis in a crustacean. (Paper IV) In a previous study (Lin et al., 2011), which aimed to find genes that are affected by the addition of Ast1 using a suppression subtractive hybridization (SSH), a protein named crustacean hematopoietic factor (CHF) was identified. In addition to CHF, several β-thymosin-like sequences were also upregulated. The β-thymosins are actin-binding proteins that have been re29 ported to participate in many cellular activities in vertebrates such as cell migration, morphogenesis and cell cycle. Until now, at least 15 β-thymosins have been identified from both vertebrates and invertebrates; thymosin-β4 is the most abundant β-thymosin in mammals (Sribenja et al. 2013). In this study, five β-thymosins were identified in freshwater crayfish and named as Pl-β-thymosin 1-5 (GenBank Accession number JX272322 – JX272325 for Pl-β-thymosin1-4, and KC460336 for Pl-β-thymosin5). Pl-β-thymosin 1 and 2 were chosen for further studies because they exhibit high expression in the brain, HPT and hemocytes. These two transcripts consist of single Tβ domains, as is the case with most vertebrate β-thymosins (Mannherz and Hannappel, 2009). However, these two Pl-β-thymosins differ in length and in the sequence of the Tβ domain C-terminal helix, which was previously shown to be important for the actin-binding function of this protein (Hertzog et al, 2004). Similar to human Tβ4, Pl-β-thymosins have no signal peptide, but at least one Pl-β-thymosin could be detected in the culture medium when crayfish HPT cells were treated with Ast1 in the present study. Human Tβ4 can interact with the β-subunit of ATP synthase on human vein endothelial cell membranes (HUVEC), causing the induction of extracellular ATP production (Freeman et al., 2011). In this study, Pl-β-thymosin 1 and 2 were shown to bind to the β-subunit of ATP synthase, as is the case with human Tβ4. However, an increase in extracellular ATP synthesis was observed only when the HPT cells were incubated with recombinant Pl-β-thymosin 1; this effect is opposite to the effect of Ast1 that caused the reduction of ATP production (Lin et al. 2009). Moreover, the study in HUVECs demonstrates that the binding of Tβ4 to the cell membrane ATP synthase is important for HUVEC migration (Freeman et al., 2011). In this study, the treatment of Pl-βthymosin 1 promoted HPT cell migration in vitro; this effect was inhibited by Ast1. In contrast, Pl-β-thymosin 2 alone had no effect on HPT cell migration, but migration increased in the presence of Ast1. However, both Pl-βthymosins transiently increased semigranular hemocyte numbers in vivo. In mammals, Tβ4 has also been reported to cause the reduction of intracellular ROS in cardiac fibroblasts (Kumar and Gupta, 2011). In crayfish, in vivo injection of Pl-β-thymosin1 had a similar effect in decreasing ROS levels in the APC, but Pl-β-thymosin 2 exhibited opposite effect. In agreement with the in vitro study, knockdown of Pl-β-thymosin 1 resulted in the reduction of SOD expression. The higher ROS level in APC, lower circulating hemocyte number and higher HPT cell proliferation in the previous study were shown to be a consequence of LPS or laminarin injection (Paper III). In addition, in an earlier study, higher plasma Ast1 levels were detected after LPS injection (Söderhäll et al., 2005). In this study, intracellular Pl-βthymosin levels increased in hemocytes and HPT in response to LPS injection. 30 Therefore, these data indicate that the balance between the Pl-βthymosins and Ast1 is of importance in regulating crayfish hemocyte homeostasis. The different effects of Pl-β-thymosin 1 and Pl-β-thymosin 2 are most likely due to differences in their structure. However, these diverse effects indicate that these proteins are involved in a complex interaction that regulates hematopoietic stem cell proliferation and differentiation. Circadian regulation of melanization and prokineticin homologues is conserved in the brain of freshwater crayfish and zebrafish. (Paper V) The SCN in the hypothalamus is a center of the mammalian circadian clock that regulates the circadian oscillation of peripheral tissues and behavior by secreting a peptide called prokineticin 2 (Prok2), which acts as an intermediate between the central nervous system and peripheral tissues (Cheng et al., 2002, Prosser et al., 2007). Transcription of Prok2 is regulated by the E-box element in its promoter region, while this element is not found in the Prok1 gene promoter region (Cheng et al., 2002, Cheng et al., 2005, Negri et al., 2007). In rodents, it has been shown that rhythmic expression of Prok2 in SCN is regulated by an endogenous circadian clock and external light. In non-mammalian vertebrates such as zebrafish, the expression of Prok2 was found in many areas of the adult brain, and it has also been reported to be involved in neural repair in adult zebrafish brain (Ayari et al., 2010). However, there is no report of circadian variation of Proks in this animal, although other components of the circadian clock have been identified and shown to be expressed from the early stages of development. The rhythmic expression of these components requires external light, which activates photoreceptor cells in the fish pineal gland (Dekens and Whitmore et al., 2008, Vatine et al., 2011). Bright light adaptation of zebrafish has been reported to require tyrosinase enzyme function in the RPE surrounding the photoreceptor cells to produce L-DOPA, a precursor of dopamine. Like Prok2, the promoter region of the tyrosinase gene also contains an E-box motif. There are five E-box motifs found in the tyrosinase gene promoter region (Camp et al., 2003), suggesting that this gene is under circadian regulation similar to that which controls Prok2 gene. In this study, the expression profiles of Prok1 and Prok2 as well as tyrosinase in adult zebrafish brain were examined at different time points during the day. The results demonstrate that these three genes exhibited a circadian expression pattern. The expression of Prok1 was increased at dawn and maintained at high levels during the day but low levels at night. The Prok2 levels also increased at dawn, but the levels remained high for a few hours only in the morning. Interestingly, the expression of tyrosinase increased 31 right after the elevation of Prok2 levels and had a profile similar to that of Prok2. Moreover, the rhythmic expression of prokineticin homologs in crayfish Ast1 and Ast2 and the invertebrate melanization enzyme proPO was also found in the adult crayfish brain. Ast1, which is more closely related to Prok2 than Ast2 (Lin, et al, 2010), has rhythmic expression similar to zebrafish Prok2, but Ast2 has the highest expression during the night. This rhythmic expression of Ast1 and Ast2 was also found in crayfish hemocytes and HPT (Wattanasurorot et al., 2011b). As is the case with tyrosinase and Prok2, proPO exhibited a similar expression pattern to Ast1. This result indicates that the circadian regulation of these proteins is conserved among different animal species. In vertebrates, it is known that there are two origins of tyrosinaseexpressing pigment cells: 1) melanocytes or melanophores, which are of neural crest origin, and 2) RPE, which is of optic cup origin. However, both of them are of neural origin (Murisier and Beermann, 2006). In this study, tyrosinase was found to have high expression in neuronal cells in adult zebrafish telencephalon. In contrast to fish tyrosinase, PO activity was found only in hemocytes that infiltrated into the brain and not in the brain cells of adult crayfish. This result suggests that crayfish hemocytes are most likely closely related to neurons. This hypothesis is in agreement with recent studies that reported that crayfish neuroprogenitors are from another tissue; HPT cells, progenitors of crayfish hemocytes, were proposed to be an external source of neuroprogenitors (Beltz et al., 2011; Benton et al., 2011). 32 Concluding remarks In this study, which aimed to gain a better understanding of the regulation of the proPO activating system and melanization and maintenance of hemocyte homeostasis in the crayfish, Pacifastacus leniusculus, a new regulator of PO activity was found to be a caspase-like-protein. Furthermore, a role for Pl-βthymosins in the regulation of hemocyte synthesis was found. In addition, evidence of a close relationship between the hematopoietic system and nervous system was also provided. From this study, it was shown that pathogenic and non-pathogenic A. hydrophila strains activate the proPO system differently. The non-pathogenic strain moderately induced PO activity, whereas the pathogenic bacteria strongly induced the system. Pathogenic bacteria might target the control system for PO activation or activity as shown by downregulation of pacifastin (Paper I) as well as the slow production of proPO-casp fragments (Paper II) after A. hydrophila infection. However, strong PO induction does not seem to be helpful for the crayfish because none survived the infection. This effect is most likely a result of the detrimental side effects of the intermediate molecules produced by the proPO activating system together with the toxin produced by bacteria. Therefore, this information indicates that tight control of the proPO system is important for maintaining the balance of fighting against pathogens without damaging the host. Another important factor for survival of the host is to maintain hemocyte production and homeostasis. Because hemocytes participate in both humoral and cellular immune responses, some hemocytes sacrifice themselves during infection to protect the host. Consequently, microbial infection causes a dramatic decrease of circulating hemocytes. The degranulation or lysis of hemocytes during infection or microbial molecule injection results in higher plasma levels of some proteins such as Ast1 (Söderhäll et al. 2005). High plasma Ast1 induces the proliferation and differentiation of the HPT cells especially in anterior part and APC (Söderhäll et al. 2005, Paper III). Higher metabolic rate of the proliferating cells induced by Ast1 possibly cause higher ROS production of cells in the APC soon after infection (Figure 6). The ROS is hypothesized to activate cell proliferation and differentiation in the anterior HPT (Paper III). In addition, plasma Ast1 most likely induces expression of Pl-β-thymosins in hemocytes and HPT as well as Pl-βthymosin release. Pl-β-thymosin 1 then counteracts Ast1 activity by decreas33 ing ROS production and increasing extracellular ATP production, and finally inducing HPT cell migration and release (Paper IV). Hemocytes are not only important for immune responses. They have recently been proposed to be an external source for neuronal precursors in crayfish (Beltz et al., 2011, Benton et al., 2011). In Paper V, proPO was shown to exhibit circadian regulation similar to the vertebrate melanization enzyme, tyrosinase. However, no brain cells were found that possessed PO activity except the hemocytes in the brain. In contrast, neural cells in the zebrafish forebrain expressed tyrosinase. These results provide another clue to the close relationship between hemocytes and the brain in crayfish as well as the crayfish hemocyte and cells with a neural origin in vertebrates. Figure 6. A scheme for regulation of hemocyte homeostasis by Ast1 and PlThymosins in crayfish. Hemocyte lysis caused by infection results in elevation of plasma Ast1 level. Ast1 then activates HPT and APC cells proliferation. Higher metabolic rate of proliferating cells probably causes escalation of ROS in the APC. The ROS may act as an additional factor that promotes cell proliferation and differentiation. In addition, Ast1 binds to membrane ATP synthase and reduces extracellular ATP production. Higher plasma Ast1 also induced Pl-thymosin expression and release. Pl-thymosin then acts as a negative regulator of Ast1 activity by reducing ROS level and stimulating ATP production of membrane ATP synthase upon binding. Induction of extracellular ATP by Pl-thymosin probably stimulates HPT cell migration. (+) represents induction. (-) represents reduction. APC, anterior proliferation center; HPT, hematopoietic tissue; ROS, reactive oxygen species; TGase, transglutaminase; THC, total hemocytes. 34 Svensk sammanfattning Ryggradslösa djur som t.ex. insekter och skaldjur har inget förvärvat immunförsvar med antikroppar utan måste helt förlita sig på medfödda system. De ryggradslösa djuren har heller inga röda blodkroppar utan i många djur transporteras syre runt till kroppens alla delar genom att binda till fritt cirkulerande syrebindande pigment. Djurens blod, som kallas hemolymfa, består av plasma och cirkulerande blodkroppar s.k. hemocyter som har stora likheter med människans vita blodkroppar. Hemocyterna är nödvändiga för att djuren ska kunna överleva, och har avgörande betydelse för immunförsvaret. Cellerna förbrukas kontinuerligt, speciellt vid infektioner och måste därför kunna nybildas snabbt. Kräftor lever i vatten och är hela tiden omgivna av mikroorganismer, och eftersom de har ett öppet cirkulationssystem måste immunförsvaret hela tiden vara i hög beredskap. När blodkroppsantalet sjunker snabbt, t.ex. vid en skada eller infektion gäller det för kräftan att snabbt få fram nya blodkroppar. Strax under skalet ovanför magen har kräftan en vävnad som kan jämföras med vår ryggmärg. Vävnaden är lik en membranomgiven mycket tunn behållare som sträcker sig runt kroppens stora artär och som framåt ansluter till djurens hjärna. Det här är kräftans blodkroppsbildande (hematopoietiska) organ och den kallas HPT. I HPT finns omogna blodkroppar och stamceller som hela tiden delar sig för att vara beredda på att frisläppa nya blodkroppar till cirkulationen. Stamcellerna i HPT delar sig mycket snabbt och samtidigt som vissa av dottercellerna går vidare och blir till funktionella hemocyter så pågår det hela tiden celldöd (apoptos) i vävnaden. När kräftan råkar ut för en skada eller infektion och mycket snabbt behöver extra tillflöde av väldigt många blodkroppar, då avtar celldöden och fler dotterceller dirigeras till att bli hemocyter. Genom odling av hematopoietiska stamceller har en ny typ av tillväxtregulator som är nödvändiga för att nya blodkroppar ska bildas i dessa djur upptäckts. Dessa proteiner, s.k. astakiner (Ast) liknar till sin struktur prokineticiner som är små regulatoriska proteiner som består av ca 80 aminosyror hos ryggradsdjur. I den här avhandlingen har den hematopoietiska vävnaden studerats i detalj, och en större utbredning av vävnaden än tidigare var känt har upptäckts. Vävnaden sträcker sig framåt i djuret och ansluter till hjärnan vi ett organ som kallas ”core frontal”. Här har vi upptäckt ett främre celldelningscentrum (APC), där cellerna har ett mer stamcelliknande utseende. Vi vet också att astakinerna regleras av ljuset och dygnsrytmen, och att denna har betydelse för blodkroppsbildning. I en jämförelse mellan kräfta och zebrafisk visade 35 det sig att expressionen av astakiner och dess motsvarighet i fisk, prokineticiner, och de enzymer vars aktivitet resulterar i melaninbildning reglerades av dygnsrytm på ett liknande sätt i båda dessa djur. I kräfta orsakas melaninbildning (brunfärgning) av enzymet phenoloxidas (PO) som föreligger i inaktiv form som ett proenzym (proPO) och aktiveras vid en infektion av en komplicerad kaskad av proteolytiska enzymer. ProPO finns i blodkropparna och framförallt i de granulära cellerna (GC), och har stor betydelse för kräftans försvar mot bakterier. I ett arbete användes flera olika stammar av en Gram-negativ patogen bakterie (Aeromonas hydrophila) med mutationer som orsakat variationer i cellväggsstrukturen hos denna bakterie. Det visade sig att cellväggen sammansättning var av stor betydelse för bakteriens virulens och beroende på cellväggens struktur aktiverades proPO i olika hög grad. Resultaten blev desamma för reaktionen i kräfta som när mjölbaggslarver (Tenebrio molitor) användes, vilket tyder på en likartad funktion hos proPO-aktiverande systemet hos dessa båda evertebrater. ProPO aktiveras av ett serinproteas kallat ppA, och aktiveringen resulterar i en klyvning i den N-terminala delen som resulterar i aktivt PO och en peptid med storleken ca 20 kDa. I ett arbete visades att denna peptid har kraftigt antimikrobiell aktivitet och kan klumpa ihop bakterier och döda dessa. Eftersom en kraftig melanisering, d.v.s. ett aktiverat proPO-system, inte bara är ett sätt för värddjuret att begränsa en infektion utan också kan utgöra en fara för värddjuret krävs att detta system är väl reglerat. Tidigare har flere regulatoriska proteasinhibitorer karakteriserats, men här har vi visat att det finns ytterligare en säkerhetsnivå för att undvika överaktivering av systemet. Vi fann att proPO i samband med aktivering också klyvs av ett kaspase-1 liknande enzym och därmed förlorar sin PO-aktivitet. Klyvningen åstadkom två N-terminala fragment som dessutom visade sig ha effekt i djuret så att bakteriehalten i blodet minskade. Detta är en tidigare okänt mekanism för att reglera och begränsa melanisering lokalt i djuret och visar att det är nödvändigt att hindra att detta enzym från att bild syreradikaler där de inte skall bildas utan endast vid bekämpning av infektioner och att färga och förstärka skalet. 36 Acknowledgements I would like to thank: Dr. Irene Söderhäll, my supervisor, for your supports, advises and always helping me to find the solutions for many mistakes I made. Thank you for being patient with me and for making my life here easier. Prof. Kenneth Söderhäll for giving me an opportunity to work here and to complete my PhD study. Thank you for being patient with stubborn and lazy student like me. Thank you for the all experiences that I will never forget. It was very tough but you were right it made me learned and improved a little bit at least. Dr. Pikul Jiravanichpaisal for suggestion and teaching me many techniques. Dr. Maria Lind Karlberg and Dr. Lage Cerenius for suggestions and comments during seminars. Ragnar Ajaxon for teaching me how to handle the crayfish. Dr. Xionghui Lin, for discussion, suggestions and all help. Thank you for teaching me how to do HPT cell culture and other techniques. You are a good teacher. I really appreciate your kindness, sincerity and your concern. You are a great person. Apiruck Watthanasurorot for a friendship, many help and for being my badminton partner during the first two years. Sucho Donpudsa for your humor that made me laugh a lot during the time that you were here. Dr. Netnapa Saelee, and Dr. Benjams Noopan for being very nice friends and for your sincerity. It was really nice spending time with both of you. Kingkamon Junkunlo for drawing pictures for my thesis and for being a good friend. 37 Susanne Trombley and Judith Habicher for sharing your teaching experiences. Katarina Holmborn Garpenstrand for helping me on zebrafish work. Former and present members in the lab: Miti Jearaphunt, Dr. Christoph Mentzendorf, Dr. Chenglin Wu, Dr. Enen Guo, Dr. Veronica Chico, Qin Peng, and Gizem Korkut. My best friend in Thailand, Sunida Tiamyuyen. My parents and sisters, the most important people in my life, for their great mental support and encouragement. Without their support I might have given up three years ago. 38 References Akinbowale A, Peng H, Grant P, Barton M. (2007). Antibiotic and heavy metal resistance in motile aeromonads and pseudomonads from rainbow trout (Oncorhynchus mykiss) farms in Australia. Int J Antimicrob Agents. 30, 177-182. An C, Hiromasa Y, Zhang X, Lovell S, Zolkiewski M, Tomich JM, Michel K. (2012). Biochemical characterization of Anopheles gambiae SRPN6, a malaria parasite invasion marker in mosquitoes. PLoS One. 2012;7(11):e48689. Anthong P, Watthanasurorot A, Klinbunga S, Ruangdej U, Söderhäll I, Jiravanichpaisal P. (2010). Cloning and characterization of a melanization inhibition protein (PmMIP) of the black tiger shrimp, Penaeus monodon. Fish Shellfish Immunol. 29, 464-468. Ayari B, El Hachimi KH, Yanicostas C, Landoulsi A, Soussi-Yanicostas N. (2010). Prokineticin 2 expression is associated with neural repair of injured adult zebrafish telencephalon. J Neurotrauma. 27, 959-72. Aspán A, Hall M, Söderhäll K. (1990). The effect of endogeneous proteinase inhibitors on the prophenoloxidase activating enzyme, a serine proteinase from crayfish haemocytes. Insect Biochem. 20, 485–492. Aspán A, Huang TS, Cerenius L, Söderhäll K. (1995). cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation. Proc Natl Acad Sci U S A. 92, 939-43. Becker C, Watson AJ, Neurath MF. (2013). Complex roles of caspases in the pathogenesis of inflammatory bowel disease. Gastroenterology. 144, 283-93. Belkin AM. (2011). Extracellular TG2: emerging functions and regulation. FEBS J. 278, 4704-16. Beltz BS, Zhang Y, Benton JL, Sandeman DC. (2011). Adult neurogenesis in the decapod crustacean brain: a hematopoietic connection?. Eur. J Neurosci. 34, 870-883. Benton JL, Zhang Y, Kirkhart CR, Sandeman DC, Beltz BS. (2011). Primary neuronal precursors in adult crayfish brain: replenishment from a nonneuronal source. BMC Neurosci. 12, 53. DOI:10.1186/1471-2202-1253. Camp E, Badhwar P, Mann GJ, Lardelli M. (2003). Expression analysis of a Tyrosinase promoter sequence in zebrafish. Pigment Cell Res. 16, 11726. 39 Cerenius L, Jiravanichpaisal P, Liu H, Söderhäll I. (2010a). Crustacean immunity. Adv Exp Med Biol. 708, 239-259. Cerenius L, Kawabata S, Lee BL, Nonaka M, Söderhäll K. (2010b). Proteolytic cascades and their involvement in invertebrate immunity. Trends Biochem Sci. 35, 575-583. Cerenius L, Lee BL, Söderhäll K. (2008). The proPO-system: pros and cons for its role in invertebrate immunity. Trends Immunol. 29, 263-271. Chaga O, Lignell M, Söderhäll K. (1995). The haemopoietic cells of the freshwater crayfish, Pacifastacus leniusculus. Anim Biol. 4, 59-70. Chang YT, Lin CY, Tsai CY, Siva VS, Chu CY, Tsai HJ, Song YL. (2013). The New Face of the Old Molecules: Crustin Pm4 and Transglutaminase Type I Serving as RNPs Down-Regulate Astakine-Mediated Hematopoiesis. PLoS One. 8(8):e72793. doi: 10.1371/journal.pone.0072793. Chaves da Silva PG, Benton JL, Sandeman DC, Beltz BS. (2013). Adult neurogenesis in the crayfish brain: the hematopoietic anterior proliferation center has direct access to the brain and stem cell niche. Stem Cells Dev. 22, 1027-41. Cheng MY, Bittman EL, Hattar S, Zhou QY. (2005). Regulation of prokineticin 2 expression by light and the circadian clock. BMC Neurosci. 6:17. Cheng MY, Bullock CM, Li C, Lee AG, Bermak JC, Belluzzi J, Weaver DR, Leslie FM, Zhou QY. (2002). Prokineticin 2 transmits the behavioural circadian rhythm of the suprachiasmatic nucleus. Nature. 417, 405-10. Dekens MP, Whitmore D. (2008). Autonomous onset of the circadian clock in the zebrafish embryo. EMBO J. 27, 2757-65. Dragojlovic-Munther M, Martinez-Agosto JA. (2013). Extracellular matrixmodulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function. Dev Biol. doi:pii: S0012-1606(13)00173-5. 10.1016/j.ydbio.2013.04.004. [Epub ahead of print]. Freeman KW, Bowman BR, Zetter BR. (2011). Regenerative protein thymosin beta-4 is a novel regulator of purinergic signaling. FASEB J. 25, 907-15. Giudice A, Caraglia M, Marra M, Montella M, Maurea N, Abbruzzese A, Arra C. (2010). Circadian rhythms, adrenergic hormones and trafficking of hematopoietic stem cells. Expert Opin Ther Targets. 14, 567-575. Grigorian M, Hartenstein V. (2013). Hematopoiesis and hematopoietic organs in arthropods. Dev Genes Evol. 223, 103-15. Grigorian M, Liu T, Banerjee U, Hartenstein V. (2013). The proteoglycan Trol controls the architecture of the extracellular matrix and balances proliferation and differentiation of blood progenitors in the Drosophila lymph gland. Dev Biol. doi:pii: S0012-1606(13)00136-X. 10.1016/j.ydbio.2013.03.007. [Epub ahead of print] 40 Grigorian M, Mandal L, Hartenstein V. (2011). Hematopoiesis at the onset of metamorphosis: terminal differentiation and dissociation of the Drosophila lymph gland. Dev Genes Evol. 221, 121-31. Grønskov K, Ek J, Brondum-Nielsen K. (2007). Oculocutaneous albinism. Orphanet J Rare Dis. 2:43. doi:10.1186/1750-1172-2-43 Gupta S, Kumar S, Sopko N, Qin Y, Wei C, Kim IK. (2012). Thymosin β4 and cardiac protection: implication in inflammation and fibrosis. Ann N Y Acad Sci. 1269, 84-91. Haine ER, Moret Y, Siva-Jothy MT, Rolff J. (2008). Antimicrobial defense and persistent infection in insects. Science. 322, 1257-1259. Hartenstein V. (2006). Blood cells and blood cell development in the animal kingdom. Annu Rev Cell Dev Biol. 22, 677-712. Hearing VJ. Determination of melanin synthetic pathways. (2011). J Invest Dermatol. 13, 1E8-E11. Hergenhahn HG, Aspán A, Söderhäll K. (1987). Purification and characterization of a high-Mr proteinase inhibitor of pro-phenol oxidase activation from crayfish plasma. Biochem J. 248, 223–228. Hertzog M, van Heijenoort C, Didry D, Gaudier M, Coutant J, Gigant B, Didelot G, Préat T, Knossow M, Guittet E, Carlier MF. (2004). The beta-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly. Cell. 117, 611-23. Hillyer JF. Mosquito immunity. (2010). Adv Exp Med Biol. 708, 218-238. Hsiao CY, Song YL. (2010). A long form of shrimp astakine transcript: molecular cloning, characterization and functional elucidation in promoting hematopoiesis. Fish Shellfish Immunol. 28, 77-86. Ito K, Hirao A, Arai F, Takubo K, Matsuoka S, Miyamoto K, Ohmura M, Naka K, Hosokawa K, Ikeda Y, Suda T. (2006). Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat Med. 12, 446-451. Iwasaki A, Medzhitov R. (2010). Regulation of adaptive immunity by the innate immune system. Science. 327, 291-295. Janeway CA, Medzhitov R. (2002). Innate immune recognition. Annu Rev Immunol. 20, 197-216. Jiang H, Vilcinskas A, Kanost MR. (2010). Immunity in lepidopteran insects. Adv Exp Med Biol. 708, 181-204. Jiang R, Kim EH, Gong JH, Kwon HM, Kim CH, Ryu KH, Park JW, Kurokawa K, Zhang J, Gubb D, Lee BL. (2009). Three pairs of proteaseserpin complexes cooperatively regulate the insect innate immune responses. J Biol Chem. 284, 35652-8. Jiménez N, Canals R, Lacasta A, Kondakova AN, Lindner B, Knirel YA, Merino S, Regué M, Tomás JM. (2008). Molecular analysis of three Aeromonas hydrophila AH-3 (serotype O34) lipopolysaccharide core biosynthesis gene clusters. J Bacteriol. 190, 3176-3184. 41 Jiravanichpaisal P, Lee BL, Söderhäll K. (2006). Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization. Immunobiology. 211, 213-36. Jiravanichpaisal P, Roos S, Edsman L, Liu H, Söderhäll K. (2009). A highly virulent pathogen, Aeromonas hydrophila, from the freshwater crayfish Pacifastacus leniusculus. J Invertebr Pathol. 101, 56-66. Johansson MW, Holmblad T, Thornqvist PO, Cammarata M, Parrinello N, Söderhäll K. (1999). A cell-surface superoxide dismutase is a binding protein for peroxinectin, a cell-adhesive peroxidase in crayfish. J Cell Sci. 112, 917-925. Johansson MW, Keyser P, Sritunyalucksana K, Söderhäll K. (2000). Crustacean haemocytes and haematopoiesis. Aquaculture. 191, 45-52. Kan H, Kim CH, Kwon HM, Park JW, Roh KB, Lee H, Park BJ, Zhang R, Zhang J, Söderhäll K, Ha NC, Lee BL. (2008). Molecular control of phenoloxidase-induced melanin synthesis in an insect. J Biol Chem. 283, 25316-25323. Keller M, Rüegg A, Werner S, Beer HD. (2008). Active caspase-1 is a regulator of unconventional protein secretion. Cell. 132(5):818-31. Koizumi N, Imamura M, Kadotani T, Yaoi K, Iwahana H, Sato R. (1999). The lipopolysaccharide-binding protein participating in hemocyte nodule formation in the silkworm Bombyx mori is a novel member of the C-type lectin superfamily with two different tandem carbohydraterecognition domains. FEBs Lett. 443, 139-143. Kollet O, Dar A, Shivtiel S, Kalinkovich A, Lapid K, Sztainberg Y, Tesio M, Samstein RM, Goichberg P, Spiegel A, Elson A, Lapidot T. (2006). Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells. Nat Med. 12, 657-664. Krzemien J, Oyallon J, Crozatier M, Vincent A. (2010). Hematopoietic progenitors and hemocyte lineages in the Drosophila lymph gland. Dev Biol. 346, 310-319. Kumar S, Gupta S. (2011). Thymosin beta 4 prevents oxidative stress by targeting antioxidant and anti-apoptotic genes in cardiac fibroblasts. PLoS One. 6(10):e26912. Lebestky T, Chang T, Hartenstein V, Banerjee U. (2000). Specification of Drosophila hematopoietic lineage by conserved transcription factors. Science. 288, 146-9. Lee JE, Edery I. (2008). Circadian regulation in the ability of Drosophila to combat pathogenic infections. Curr Biol. 18, 195-9. Lee H, Kwon HM, Park JW, Kurokawa K, Lee BL. (2009). N-terminal GNBP homology domain of Gram-negative binding protein 3 functions as a beta-1,3-glucan binding motif in Tenebrio molitor. BMB Rep. 42, 506-510. 42 Lee SY, Wang R, Söderhäll K. (2000). A lipopolysaccharide- and β-1,3glucan-binding protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus. J Biol Chem. 275, 1337-1343. Lewandowski D, Barroca V, Ducongé F, Bayer J, Nhieu JTV, Pestourie C, Fouchet P, Tavitian B, Roméo PH. (2010). In vivo cellular imaging pinpoints the role of reactive oxygen species in the early steps of adult hematopoietic reconstitution. Blood. 115, 443-452. Li JD, Hu WP, Zhou QY. (2012). The circadian output signals from the suprachiasmatic nuclei. Prog Brain Res. 199, 119-27. Liang Z, Lindblad P, Beauvais A, Johansson MW., Latgé JP, Hall M, Cerenius L, Söderhäll K. (1992). Crayfish α-macroglobulin and 76 kDa protein; Their biosynthesis and subcellular localization of the 76 kDa protein. I Insect Physiol. 38. 987-995. Liang Z, Sottrup-Jensen L, Aspán A, Hall M, Söderhäll K. (1997). Pacifastin, a novel 155-kDa heterodimeric proteinase inhibitor containing a unique transferrin chain. Proc Natl Acad Sci USA. 94, 6682-6687. Lin X, Cerenius L, Lee BL, Söderhäll K. (2007). Purification of properoxinectin, a myeloperoxidase homologue and its activation to a cell adhesion molecule. Biochim Biophys Acta. 1770, 87-93. Lin X, Kim YA, Lee BL, Söderhäll K, Söderhäll I. (2009). Identification and properties of a receptor for the invertebrate cytokine astakine, involved in hematopoiesis. Exp Cell Res. 315, 1171-80. Lin X, Novotny M, Söderhäll K, Söderhäll I. (2010). Ancient cytokines, the role of astakines as hematopoietic growth factors. J Biol Chem. 285, 28577-28586. Lin X, Söderhäll I. (2011). Crustacean hematopoiesis and the astakine cytokines. Blood. 117, 6417-6424. Lin X, Söderhäll K, Söderhäll I. (2011). Invertebrate hematopoiesis: an astakine-dependent novel hematopoietic factor. J Immunol. 186, 20732079. Lin X, Söderhäll K, Söderhäll I. (2008). Transglutaminase activity in the hematopoietic tissue of a crustacean, Pacifastacus leniusculus, importance in hemocyte homeostasis. BMC Immunol. 9, 58 doi:10.1186/1471-2172-9-58 Ling E, Yu XQ. (2006). Cellular encapsulation and melanization are enhanced by immulectins, pattern recognition receptors from the tobacco hornworm Manduca sexta. Dev Comp Immunol. 30, 289-299. Liu H, Jiravanichpaisal P, Cerenius L, Lee BL, Söderhäll I, Söderhäll K. (2007). Phenoloxidase is an important component of the defense against Aeromonas hydrophila infection in a crustacean, Pacifastacus leniusculus. J Biol Chem. 282, 33593-33598. 43 Liu H, Wu C, Matsuda Y, Kawabata S, Lee BL, Söderhäll K, Söderhäll I. (2011). Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)?. Dev Comp Immunol. 35, 51-61. Makhijani K, Alexander B, Tanaka T, Rulifson E, Brücker K. (2011). The peripheral nervous system supports blood cell homing and survival in the Drosophila larva. Development. 138, 5379-5391. Mannherz HG, Hannappel E. (2009). The beta-thymosins: intracellular and extracellular activities of a versatile actin binding protein family. Cell Motil Cytoskeleton. 66, 839-51. Martin SJ, Henry CM, Cullen SP. (2012). A perspective on mammalian caspases as positive and negative regulators of inflammation. Mol Cell. 46(4):387-97. Meister M, Govind S. (2006). Hematopoietic development in Drosophila: a parallel with vertebrates. Hematopoietic Stem Cell Development. Eds. Godin I and Cumano A. pp: 124-141. Springer US. Merino S, Camprubí S, Tomás J. (1991). The role of lipopolysaccharide in complement-killing of Aeromonas hydrophila strains of serotype O:34. J Gen Microbiol. 137, 1583-1590. Meshorer E, Misteli T. (2006). Chromatin in pluripotent embryonic stem cells and differentiation. Nat Rev Mol Cell Biol. 7, 540-546. Méndez-Ferrer S, Lucas D, Battista M, Frenette PS. (2008). Haematopoietic stem cell release is regulated by circadian oscillations. Nature. 452, 442-7. Mohyeldin A, Garzón-Muvdi T, Quiñones-Hinojosa A. (2010). Oxygen in stem cell biology: a critical component of the stem cell niche. Cell Stem Cell. 7, 150-61. Murisier F, Beermann F. (2006). Genetics of pigment cells: lessons from the tyrosinase gene family. Histol Histopathol. 21, 567-78. Nappi A, Poirié M, Carton Y. (2009). The role of melanization and cytotoxic by-products in the cellular immune responses of Drosophila against parasitic wasps. Adv Parasit. 70, 99-121. Negri L, Lattanzi R, Giannini E, Canestrelli M, Nicotra A, Melchiorri P. (2009). Bv8/Prokineticins and their Receptors A New Pronociceptive System. Int Rev Neurobiol. 85, 145-57. Negri L, Lattanzi R, Giannini E, Melchiorri P. (2007). Bv8/Prokineticin proteins and their receptors. Life Sci. 81(14):1103-16. Nickel W, Rabouille C. (2009). Mechanisms of regulated unconventional protein secretion. Nat Rev Mol Cell Biol. 10, 148-55. Nogueras M, Merino S, Aguilar A, Benedi V, Tomás J. (2000). Cloning, sequencing, and role in serum susceptibility of porin II from mesophilic Aeromonas hydrophila. Infect Immun. 68, 1849-1854. Owusu-Ansah E, Banerjee U. (2009). Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation. Nature. 461, 537-541. 44 Page-McCaw PS, Chung SC, Muto A, Roeser T, Staub W, Finger-Baier KC, Korenbrot JI, Baier H. (2004). Retinal network adaptation to bright light requires tyrosinase. Nat Neurosci. 7, 1329-36. Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, Schultz PG, Kay SA, Takahashi JS, Hogenesch JB. (2002). Coordinated transcription of key pathways in the mouse by the circadian clock. Cell. 109, 307-20. Park JW, Kim CH, Rui J, Park KH, Ryu KH, Chai JH, Hwang HO, Kurokawa K, Ha NC, Söderhill I, Söderhill K, Lee BL. (2010). Beetle immunity. Adv Exp Med Biol. 708, 163-80. Parker JL, Shaw JG. (2011). Aeromonas spp. Clinical biology and disease. J Infect. 2011 Feb;62(2):109-18. Pasupuleti M, Davoudi M, Malmsten M, Schmidtchen A. (2009). Antimicrobial activity of a C-terminal peptide from human extracellular superoxide dismutase. BMC Res Notes. 2:136. doi: 10.1186/1756-0500-2136. Prosser HM, Bradley A, Chesham JE, Ebling FJ, Hastings MH, Maywood ES. (2007). Prokineticin receptor 2 (Prokr2) is essential for the regulation of circadian behavior by the suprachiasmatic nuclei. Proc Natl Acad Sci U S A. 104, 648-53. Rabaan A, Gryllos I, Tomás J, Shaw J. (2001). Motility and the polar flagellum are required for Aeromonas caviae adherence to HEp-2 cells. Infect Immun. 69, 4257-4267. Roulston C, Smith VJ. (2011). Isolation and in vitro characterisation of prohaemocytes from the spider crab, Hyas araneus (L.). Dev Comp Immunol. 35, 537-44. Sahoo PK, Mahapatra KD, Saha JN, Barat A, Sahoo M, Mohanty BR, Gjerde B, Ødegård J, Rye M, Salte R. (2008). Family association between immune parameters and resistance to Aeromonas hydrophila infection in the Indian major carp, Labeo rohita. Fish Shellfish Immunol. 25, 163-169. Santra M, Chopp M, Zhang ZG, Lu M, Santra S, Nalani A, Santra S, Morris DC. (2012). Thymosin β 4 mediates oligodendrocyte differentiation by upregulating p38 MAPK. Glia. 60, 1826-38. Scheiermann C, Kunisaki Y, Frenette PS. (2013). Circadian control of the immune system. Nat Rev Immunol. 13, 190-198. Schneider DS, Chambers MC. (2008). Rogue insect immunity. Science. 322, 1199-1200. Schulenburg H, Boehnisch C, Michiels NK. (2007). How do invertebrates generate a highly specific innate immune response? Mol Immunol. 44, 3338-3344. Seshadri R, Joseph S, Chopra A, Sha J, Shaw J, Graf J, Haft D, Wu M, Ren Q, Rosovitz MJ, Madupu R, Tallon L, Kim M, Jin S, Vuong H, Stine OC, Ali A, Horneman AJ, Heidelberg JF. (2006). Genome sequence of 45 Aeromonas hydrophila ATCC 7966T: Jack of all trades. J Bacteriol. 188, 8272-8282. Sha J, Pillai L, Fadl A, Galindo C, Erova T, Chopra A. (2005). The type III secretion system and cytotoxic enterotoxin alter the virulence of Aeromonas hydrophila. Infect Immun. 73, 6446-6457. Sha J, Wang SF, Suarez G, Sierra JC, Fadl AA, Erova TE, Foltz SM, Khajanchi BK, Silver A, Graf J, Schein CH, Chopra AK. (2007). Further characterization of a type III secretion system (T3SS) and of a new effector protein from a clinical isolate of Aeromonas hydrophila Part I. Microb Pathog. 43, 127-146. Shirasu-Hiza MM, Dionne MS, Pham LN, Ayres JS, Schneider DS. (2007). Interactions between circadian rhythm and immunity in Drosophila melanogaster. Curr Biol. 17, R353-355. Sierra JC, Suarez G, Sha J, Foltz SM, Popov VL, Galindo CL, Garner HR, Chopra AK. (2007). Biological characterization of a new type III secretion system effector from a clinical isolate of Aeromonas hydrophila - Part II. Microb Pathog. 43, 147-160. Sinenko SA, Shim J, Banerjee U. (2012). Oxidative stress in the haematopoietic niche regulates the cellular immune response in Drosophila. EMBO Rep. 13, 83-89. Silver AC, Arjona A, Walker WE, Fikrig E. (2012). The circadian clock controls toll-like receptor 9-mediated innate and adaptive immunity. Immunity. 36, 251-61. Slominski A, Zmijewski MA, Pawelek J. (2012). L-tyrosine and Ldihydroxyphenylalanine as hormone-like regulators of melanocyte functions. Pigment Cell Melanoma Res. 25, 14-27. Smaaland R, Sothern RB, Laerum OD, Abrahamsen JF. (2002). Rhythms in human bone marrow and blood cells. Chronobiol Int. 19, 101-127. Smith VJ, Söderhäll K. (1983a). β-1,3 glucan activation of crustacean hemocytes in vitro and in vivo. Biol Bull. 164, 299-314 Smith VJ, Söderhäll K. (1983b). Induction of degranulation and lysis of haemocytes in the freshwater crayfish, Astacus astacus by components of the prophenoloxidase activating system in vitro. Cell Tissue Res. 233, 295-303. Sollberger G, Strittmatter GE, Garstkiewicz M, Sand J, Beer HD. (2013). Caspase-1: The inflammasome and beyond. Innate Immun. [Epub ahead of print] Spiegel A, Kalinkovich A, Shivtiel S, Kollet O, Lapidot T. (2008). Stem cell regulation via dynamic interactions of the nervous and immune systems with the microenvironment. Cell Stem Cell. 3, 484-492. Sribenja S, Wongkham S, Wongkham C, Yao Q, Chen C. (2013). Roles and mechanisms of β-thymosins in cell migration and cancer metastasis: an update. Cancer Invest. 31, 103-10. 46 Stone EF, Fulton BO, Ayres JS, Pham LN, Ziauddin J, Shirasu-Hiza MM. (2012). The circadian clock protein timeless regulates phagocytosis of bacteria in Drosophila. PLoS Pathog. 8(1):e1002445. Storch KF, Lipan O, Leykin I, Viswanathan N, Davis FC, Wong WH, Weitz CJ. (2002). Extensive and divergent circadian gene expression in liver and heart. Nature. 417, 78-83. Suarez G, Sierra JC, Sha J, Wang S, Erova TE, Fadl AA, Foltz SM, Horneman AJ, Chopra AK. (2008). Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila. Microb Pathog. 44, 344-361. Söderhäll I, Bangyeekhun E, Mayo S, Söderhäll K. (2003). Hemocyte production and maturation in an invertebrate animal; proliferation and gene expression in hematopoietic stem cells of Pacifastacus leniusculus. Dev Comp Immunol. 27, 661-672. Söderhäll I, Kim YA, Jiravanichpaisal P, Lee SY, Söderhäll K. (2005). An ancient role for a prokineticin domain in invertebrate hematopoiesis. J Immunol. 174, 6153-6160. Söderhäll I, Wu C, Novotny M, Lee BL, Söderhäll K. (2009). A novel protein acts as a negative regulator of prophenoloxidase activation and melanization in the freshwater crayfish Pacifastacus leniusculus. J Biol Chem. 284, 6301-6310. Söderhäll K, Smith VJ. (1983). Separation of the haemocyte populations of Carcinus maenas and other marine decapods, and prophenoloxidase distribution. Dev Comp Immunol. 7, 229-39. Takizawa H, Boettcher S, Manz MG. (2012). Demand-adapted regulation of early hematopoiesis in infection and inflammation. Blood. 119, 29913002. Vatine G, Vallone D, Gothilf Y, Foulkes NS. (2011). It's time to swim! Zebrafish and the circadian clock. FEBS Lett. 585, 1485-94. Vladimer GI, Marty-Roix R, Ghosh S, Weng D, Lien E. (2013). Inflammasomes and host defenses against bacterial infections. Curr Opin Microbiol. 2013 Feb;16(1):23-31. Wang N, Hebert DN. (2006). Tyrosinase maturation through the mammalian secretory pathway: bringing color to life. Pigment Cell Res. 19, 3-18. Wang Z, Griffin M. (2012). TG2, a novel extracellular protein with multiple functions. Amino Acids. 42, 939-49. Watthanasurorot A, Jiravanichpaisal P, Liu H, Söderhäll I, Söderhäll K. (2011a). Bacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus. PLoS Pathog. 7(6):e1002062. Watthanasurorot A, Saelee N, Phongdara A, Roytrakul S, Jiravanichpaisal P, Söderhäll K, Söderhäll I. (2013). Astakine 2--the dark knight linking melatonin to circadian regulation in crustaceans. PLoS Genet. 9(3):e1003361. 47 Watthanasurorot A, Söderhäll K, Jiravanichpaisal P, Söderhäll I. (2011b). An ancient cytokine, astakine, mediates circadian regulation of invertebrate hematopoiesis. Cell Mol Life Sci. 68, 315-323. Wilking M, Ndiaye M, Mukhtar H, Ahmad N. (2013). Circadian rhythm connections to oxidative stress: implications for human health. Antioxid Redox Signal. 19, 192-208. Wu C, Charoensapsri W, Nakamura S, Tassanakajon A, Söderhäll I, Söderhäll K. (2013). An MBL-like protein may interfere with the activation of the proPO-system, an important innate immune reaction in invertebrates. Immunobiology. 218, 159-68. Wu C, Söderhäll I, Kim YA, Liu H, Söderhäll K. (2008). Hemocyte-lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus. Proteomics. 8, 4226-4235. Wu C, Söderhäll K, Söderhäll I. (2011). Two novel ficolin-like proteins act as pattern recognition receptors for invading pathogens in the freshwater crayfish Pacifastacus leniusculus. Proteomics. 11:2249-2264. Yu XQ, Kanost MR. (2004). Immulectin-2, a pattern recognition receptor that stimulates hemocyte encapsulation and melanization in the tobacco hornworm, Manduca sexta. Dev Comp Immunol. 28, 891-900. Zhao M, Söderhäll I, Park JW, Ma YG, Osaki T, Ha NC, Wu CF, Söderhäll K, Lee BL. (2005). A novel 43-kDa protein as a negative regulatory component of phenoloxidase-induced melanin synthesis. J Biol Chem. 280, 24744-24751. 48 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1086 Editor: The Dean of the Faculty of Science and Technology A doctoral dissertation from the Faculty of Science and Technology, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology. 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