Neuro-immune lessons from an annelid: The medicinal leech

Article (PDF Available)inDevelopmental and comparative immunology 66 · July 2016with 139 Reads
DOI: 10.1016/j.dci.2016.06.026
Abstract
An important question that remains unanswered is how the vertebrate neuroimmune system can be both friend and foe to the damaged nervous tissue. Some of the difficulty in obtaining responses in mammals probably lies in the conflation in the central nervous system (CNS), of the innate and adaptive immune responses, which makes the vertebrate neuroimmune response quite complex and difficult to dissect. An alternative strategy for understanding the relation between neural immunity and neural repair is to study an animal devoid of adaptive immunity and whose CNS is well described and regeneration competent. The medicinal leech offers such opportunity. If the nerve cord of this annelid is crushed or partially cut, axons grow across the lesion and conduction of signals through the damaged region is restored within a few days, even when the nerve cord is removed from the animal and maintained in culture. When the mammalian spinal cord is injured, regeneration of normal connections is more or less successful and implies multiple events that still remain difficult to resolve. Interestingly, the regenerative process of the leech lesioned nerve cord is even more successful under septic than under sterile conditions suggesting that a controlled initiation of an infectious response may be a critical event for the regeneration of normal CNS functions in the leech. Here are reviewed and discussed data explaining how the leech nerve cord sensu stricto (i.e. excluding microglia and infiltrated blood cells) recognizes and responds to microbes and mechanical damages.
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Figure legends 692 693 Figure 1: (a) Schematic cross section of H. medicinalis' body. The CNS is enclosed within the 694 ventral blood sinus. (b) Defined amount of bacteria (mix of heat-killed M. nishino: Micrococcus 695 nishinomiyaensis and A. hydrophila: Aeromonas hydrophila at 3x10 7 CFU/ml) promotes the 696 regeneration process relative to sterile conditions (Schikorski, 2008). The Gram-positive and 697 Gram-negative bacteria M. nishino and A. hydrophila, respectively, were isolated from the 698 natural environment of Hirudo. Sectioning of one side of the paired connective nerve linking 699 adjacent segmental ganglia was performed on excised nerve cords maintained in culture. To 700 monitor the progress of nerve repair, micrographs of the damaged nerve cords were taken every 701 24 h, in the presence or absence of bacteria. Under sterile conditions, as documented in the left 702 column, restoration of the connective nerve across the cut begins at ~4 days post-axotomy (panel 703 J 4) and is finished 4 days later. In comparison, nerve repair is evident sooner in the presence of 704 a controlled number of bacteria, reconnection starting after 2 days or 3 days (right column). 705 706 Figure 2: Modulation of the gene expression of leech PRRs and immune effectors in nerve cords 707 incubated for 6 hours with various microbial components (2 µg/ml of LTA: lipoteichoïc acid; 10 708 µg/ml of MDP: muramyl dipeptide; 100 ng/ml of LPS: lipopolysaccharides), heat killed bacteria 709 at a concentration favoring the regenerative process (see Fig. 1b); VSV: Vesicular stomatitis 710 virus or viral mimetic (10 µg/ml of polyI:C). Incubations without microbial components or 711 bacteria were performed in the same conditions as controls. For the details see: Cuvillier-Hot et 712 al., 2011; Schikorski et al.,2008; Schikorski et al., 2009. 713 714 Figure 3: TLR pathways in the leech CNS. Analysis of the Hirudo transcriptome database 715 reveals the presence of putative homologs of nearly all factors reported to play critical roles in 716 human TLR pathways. Framed E values give the homologies of the leech proteins with their 717 counterparts identified in Ce : Caenorhabditis elegans, Dm : Drosophila melanogaster and Hs : 718 Homo sapiens. Some of them (E values 0) has already been entirely characterized. 719 720 Figure 4: The level of AMP expression is enhanced in the CNS of leeches that have swum for 6 721 hours in water heavily enriched in a mix of alive bacteria (A. nishinomyaensis and A. hydrophila 722
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Accepted Manuscript
Neuro-immune lessons from an annelid: The medicinal leech
Aurélie Tasiemski, Michel Salzet
PII: S0145-305X(16)30216-6
DOI: 10.1016/j.dci.2016.06.026
Reference: DCI 2675
To appear in: Developmental and Comparative Immunology
Received Date: 21 April 2016
Revised Date: 9 June 2016
Accepted Date: 30 June 2016
Please cite this article as: Tasiemski, A., Salzet, M., Neuro-immune lessons from an annelid: The
medicinal leech, Developmental and Comparative Immunology (2016), doi: 10.1016/j.dci.2016.06.026.
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Neuro-immune lessons from an annelid: the medicinal leech 1
2
Aurélie Tasiemski
a*
and Michel Salzet
b
3
4
a
Université de Lille, CNRS UMR8198, Unité d’Evolution, Ecologie et Paléontologie (EEP), 5
Species Interactions and Comparative Immunology (SPICI) team, 59655 Villeneuve d’Ascq, 6
France 7
b
Université de Lille, INSERM U-1192, Laboratoire de Protéomique, Réponse Inflammatoire, 8
Spectrométrie de Masse (PRISM), 59655 Villeneuve d’Ascq, France 9
10
Abstract: 11
An important question that remains unanswered is how the vertebrate neuroimmune system can 12
be both friend and foe to the damaged nervous tissue. Some of the difficulty in obtaining 13
responses in mammals probably lies in the conflation in the central nervous system (CNS), of the 14
innate and adaptive immune responses, which makes the vertebrate neuroimmune response quite 15
complex and difficult to dissect. An alternative strategy for understanding the relation between 16
neural immunity and neural repair is to study an animal devoid of adaptive immunity and whose 17
CNS is well described and regeneration competent. The medicinal leech offers such opportunity. 18
If the nerve cord of this annelid is crushed or partially cut, axons grow across the lesion and 19
conduction of signals through the damaged region is restored within a few days, even when the 20
nerve cord is removed from the animal and maintained in culture. When the mammalian spinal 21
cord is injured, regeneration of normal connections is more or less successful and implies 22
multiple events that still remain difficult to resolve. Interestingly, the regenerative process of the 23
leech lesioned nerve cord is even more successful under septic than under sterile conditions 24
suggesting that a controlled initiation of an infectious response may be a critical event for the 25
regeneration of normal CNS functions in the leech. Here are reviewed and discussed data 26
explaining how the leech nerve cord sensu stricto (i.e. excluding microglia and infiltrated blood 27
cells) recognizes and responds to microbes and mechanical damages. 28
29
Keywords: Annelid, CNS, antimicrobial peptides, neuro-immunity, sensing receptors, 30
regeneration 31
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*Corresponding author. Email: aurelie.tasiemski@univ-lille1.fr ; Website: 32
http://spici.weebly.com/ 33
34
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1. I
NTRODUCTION
35
36
Because of its multiple vital functions, it is critical that the CNS be successfully defended against 37
pathogens. For a long time, this organ was considered to be immunologically inert and isolated 38
from the peripheral immune system. However, it is now well established that immune 39
surveillance and inflammatory responses do occur within this compartment (Wrona et al, 2006). 40
Indeed, in response to either cerebral injury or systemic bacterial infection, the CNS launches a 41
well-organized immunological reaction that encompasses both neural components and peripheral 42
immune system cells. Within the mammalian CNS, resident glial cells, including astrocytes and 43
microglia, have been shown to initiate a characteristic innate immune response by producing and 44
releasing antimicrobial peptides (AMPs), cytokines and chemokines (Becher et al., 2000). These 45
circulating molecules promote the destruction of the invading bacteria, the permeabilization of 46
the blood brain barrier (BBB), and the recruitment of peripheral leukocytes to the CNS and the 47
activation of their effector functions, including further production of cytokines as well as 48
phagocytosis by peripheral macrophages. The specific outcome of this neuroinflammatory 49
response, which has both beneficial and detrimental aspects, depends on the context of the insult 50
and on the duration of the inflammation. On the positive side, increased immune activity rapidly 51
initiates the killing of bacteria and the removal of apoptotic cells and cellular debris, while also 52
playing an important role in neuroprotection and repair by inducing the production of 53
neurotrophic factors. In fact, several recent observations suggest that induction of regeneration of 54
normal CNS function may depend critically upon the co-initiation of an immune response 55
(Gendelman et al., 2002, 2003; Nguyen et al., 2002; Pavlov and Tracey, 2015; Russo and 56
McGavern, 2015). On the negative side, excessive and/or chronic glial reactivity, in conjunction 57
with the presence of adaptive immune cells within the CNS, can damage the CNS by inducing 58
neuronal death and by blocking axonal myelination. An important question that remains 59
unanswered is how the vertebrate neuroimmune system can be both friend and foe to the 60
damaged nervous tissue. Some of the difficulty in obtaining an answer in mammals probably lies 61
in the conflation in the CNS of the innate and adaptive immune responses, which makes the 62
vertebrate neuroimmune response quite complex and difficult to dissect. An alternative strategy 63
for understanding the relation between neural immunity and neural repair is to study an animal 64
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devoid of adaptive immunity and whose CNS is well described and regeneration competent. The 65
medicinal leech offers such opportunity. 66
In this review, we discuss the unique characteristics of the medicinal leech as model for studying 67
the immune response of the CNS. 68
69
1. T
HE MEDICINAL LEECH AS A MODEL TO STUDY THE LINK BETWEEN IMMUNITY AND
70
REGENERATION OF THE
CNS 71
72
Several features make the CNS of the medicinal leech Hirudo verbana (often wrongly referred 73
and sold in Europe as Hirudo medicinalis) particularly attractive as a model system for the 74
exploration of interactions between bacteria, the nervous and immune systems. These features 75
include simplicity, a fixed number of neurons, and consistency from animal to animal, which 76
allow the recognition, characterization and repeated study of identified neurons, at all 77
developmental stages and following specific perturbations, such as mechanical or septic trauma. 78
The leech CNS is comprised of a fixed number (Nicholls and Van Essen, 1974) of mid-body 79
segmental ganglia, plus larger "head" and tail "brains", linked to each other by longitudinal 80
nerves known as connectives. Most segmental ganglia have a complement of ~400 neurons and 8 81
giant glial cells, along with a large population of microglia. Almost all of the ~400 neurons have 82
homologs in every ganglion; a majority has been characterized morphologically as well as 83
physiologically, and for many their synaptic connectivity, neurotransmitters and roles in 84
behaviour have been determined (Muller et al., 1979; Muller and Carbonetto, 1979). Moreover, 85
by contrast with mammals, studies in the leech can be focused exclusively on the neural immune 86
response of the CNS itself. Indeed, the leech nerve cord normally lies within a ventral blood 87
sinus, but it is encapsulated by a tough fibrous sheath that may, like the mammalian BBB, limit 88
the exchange of macromolecules and cells with the blood. However, the intact CNS can be easily 89
removed from the animal and cultured in the absence of peripheral immune system components 90
and blood cells that might infiltrate the CNS after injury (Fig. 1a). 91
The most important feature is the capacity of the medicinal leech CNS to regenerate and restore 92
normal function in response to injury. If the nerve cord of this annelid is crushed or partially cut, 93
axons grow across the lesion and conduction of signals through the damaged region is restored 94
within a few days, even when the nerve cord is removed from the animal and maintained in 95
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culture. When the mammalian spinal cord is injured, regeneration of normal connections is more 96
or less successful and implies multiple events that still remain difficult to resolve. In the leech, 97
the process of regeneration begins with a rapid activation of microglial cells leading to their 98
accumulation at the lesion site (Muller and Carbonetto, 1979). Microglial cells are resident 99
macrophages in mammals (Hanisch and Kettenmann, 2007; Parry et al., 1997), arthropods 100
(Smith et al., 1987) and leeches (von Bernhardi and Muller, 1995) which respond rapidly to brain 101
injury by moving to the lesion and accumulating there. Whether they subsequently divide as in 102
mammals and arthropods, or not as in leeches, recruited microglia phagocyte cellular debris 103
(Neumann et al., 2009). Although blood cells are still in a good shape after a one week culture, 104
microglial cells die rapidly (in less than 24 hours). By developing a procedure to deplete the 105
nerve cord in microglial cells (Schikorski et al., 2008), we evidenced that an optimal 106
regeneration require microglial cells for initiation and blood cells to facilitate and accelerate the 107
process (Boidin-Wichlacz et al., 2012). 108
Interestingly, the regenerative process of the lesioned nerve cord is even more successful under 109
septic than under sterile conditions suggesting that initiation of a controlled infectious response 110
may be a critical event for the regeneration of normal CNS functions in the leech (Fig. 1b). 111
Hirudo, therefore, is an excellent model system for exploring fundamental questions about the 112
interaction of the nervous and innate immune systems, including (a) what is the nature of the 113
innate immune response mounted by the nervous system? (b) How does the nerve cord sensu 114
stricto (i.e. excluding microglia and infiltrated blood cells) respond to microbes, mechanical 115
damages and other stresses? 116
117
2. N
EURONAL MICROBIAL SENSING OF THE LEECH
118
Invertebrates, being devoid of adaptive immunity dependent on RAG (i.e. Recombination-119
Activating Genes), are interesting model systems for exploring the molecular basis of innate 120
immunity. For example, the initial evidence for the pivotal role of the Toll receptor family in 121
immunity was discovered in Drosophila, and only later in mammals (Imler and Zheng, 2004). 122
Another example is the discovery of the first antimicrobial peptides (AMPs) by Hans Boman in 123
the moth Hyalophora cecropia (Steiner et al., 1981). AMPs are now considered as important 124
effectors of the innate immune systems of both invertebrates and vertebrates. Most reports on 125
immune effectors in invertebrates have tended to focus on their involvement in the systemic anti-126
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infectious response. More and more studies described the presence of immune molecules in the 127
nervous systems of insects and nematodes, both members of the ecdysozoan group. Indeed, 128
several Toll-like receptors (TLR) and some molecules of the TLR signalling pathway have been 129
detected in glial and neuronal cells of Drosophila, and appear to have a role in neural 130
development in the larvae (Wharton and Crews, 1993). In Caenorhabditis elegans, an ortholog 131
of the Drosophila toll gene was shown to be expressed in pharyngeal neurons, where it 132
participates in defensive behaviour by discouraging the worm from ingesting pathogenic bacteria 133
(Pujol et al., 2001). Increasingly, a role for the nervous system in recognizing microbes has been 134
showed not only in the induction of host immunity but also in broader effects on host physiology 135
associated with the microbiota, the metabolism, the behavior, and the pathophysiology of 136
diseases (Kawli et al., 2010). 137
The innate immune response is an evolutionarily ancient defense strategy against pathogenic 138
agents that has been documented widely in living organisms, including invertebrates and 139
vertebrates. Its major functions include: (1) recruiting immune system cells to infection sites 140
though the production of chemokines and cytokines; (2) activating the complement cascade in 141
order to identify pathogens, activate cells to promote pathogen clearance and stimulate the 142
adaptive immune response; (3) interacting specifically with pathogens through membrane or 143
cytosolic receptors in immune circulating cells in order to remove pathogens from organs and 144
tissues. 145
To search for the major players for these functions, we screened the Hirudo transcriptome 146
database for invertebrate homologs of vertebrate genes belonging to these different categories 147
and then investigate their immune and/or neuro-regenerative functions by combining cellular, 148
molecular, biochemical and morpho-functional approaches (Macagno et al., 2010). Among the 149
various factors identified, we will lay particular emphasis on: (1) Pattern Recognition Receptors 150
(PRR) and their associated signalling pathways; (2) Immune effectors such as AMPs and 151
cytokines produced by neurons. 152
153
2.1. Pattern Recognition Receptors (PRR) expressed in the leech CNS 154
155
The innate immune system uses different PRR that sense Microbe-Associated Molecular Patterns 156
(MAMPs). These include TLRs, Retinoic-acid-inducible gene-1 (RIG-1)-Like Receptors 157
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(RLRs), and the Nod-Like Receptors (NLRs), all of which contain Leucine Rich Repeat domains 158
(LRRs). PRRs contain two key functional domains i.e. the LRR domain that interacts directly or 159
indirectly with microbial signature shared by major classes of microbes, whereas the second is 160
more like protein-protein interaction domains activating the downstream signalling event, 161
leading to the transcription of immunity effector genes such as anti-microbial substances, 162
cytokines. Such danger signaling receptors seem to be well conserved in leeches and are all 163
expressed in the CNS of the leech (Table 1). Their presence was predictable according to the 164
demonstrated ability of this organ to mount an antimicrobial response specific of the microbial 165
agent it is exposed to (Schikorski et al., 2008, 2009; Tasiemski and Salzet, 2010). 166
Toll-Like Receptors (TLRs): TLRs are critical components of the innate immune responses of 167
both vertebrates and invertebrates (Leulier et al., 2008; Imler and Hoffmann, 2000a, b, 2001; 168
Imler et al., 2000; Imler and Zheng, 2004; Tauszig et al., 2000). TLRs are type I transmembrane 169
receptors with an ectodomain containing several LRR motifs flanked by conserved cystein-rich 170
motifs, a feature they share with several other types of receptors, including GPIba, the 171
neurotrophin receptor Trk, and CD14 (Imler and Hoffmann, 2000b). Within the cytoplasm, TLRs 172
have a ~150 amino-acid domain with strong homology to a corresponding region in the Receptor 173
for Interleukin 1 (IL-1R), though the ectodomain of IL-1R has of three immunoglobulin-like 174
motifs instead of LRRs. 175
There are 10-13 known mammalian TLRs, depending on species, and many of the components 176
of the cytoplasmic pathway from TLRs to the nucleus have been identified. For example, TLR4 177
is an essential component of the lipopolysaccharide (LPS) receptor complex, together with the 178
membrane associated, GPI-linked, CD14. TLRs also mediate NF-kB activation in response to a 179
broad repertoire of microbial molecules, including the responses to (A) nucleic acids, mediated 180
by TLR3 (double-stranded (ds) RNA), TLR7 (single-stranded RNA enriched in U residues) and 181
TLR9 (unmethylated CpG motifs); (B) bacterial lipopeptides and/or fungal PAMPs, mediated by 182
the TLR2/TLR6 and TLR2/TLR1 heterodimers; and (C) bacterial proteins, e.g., TLR5 is 183
required for recognition of flagellin, whereas TLR11 mediates activation by profilin of the 184
protozoan parasite Toxoplasma gondii or protein components from uropathogenic strains of 185
Escherichia coli (Imler and Hoffmann, 2000a, 2001). 186
Among invertebrates, the innate immune response has been first and most extensively 187
investigated in ecdysozoan species: Drosophila melanogaster (De Gregorio et al., 2002; 188
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Ferrandon et al., 1998) and C. elegans (Pujol et al., 2001). A large number of TLRs are now 189
identified in various metazoan phyla: Cnidaria, Annelida, Mollusca, Arthropoda, Echinodermata 190
and Chordata (Hemmrich et al., 2007; Rauta et al., 2014). The existence of TLRs in annelids has 191
already been deduced from in silico analysis of the genomes of Capitella and Helobdella 192
(Davidson et al., 2008). As for the medicinal leech, the repertoire of Capitella consists primarily 193
of the vertebrate-like rather than a protostome like domain organisation. Interestingly, the blastp 194
homology of HmTLR1 with the vertebrate TLR13 and TLR3 is consistent with this observation. 195
In addition to the sequence homology, HmTLR1 is expressed by both microglia and neurons and 196
seems to exert functions comparable to those described for the mammalian TLR3 in the brain 197
(see above). Indeed, in vertebrates, microglia has been reported to express mRNAs for TLRs 1 to 198
9 whereas neurons and oligodendrocytes (Prehaud et al., 2005) express only transcripts encoding 199
TLR3 (Bsibsi et al., 2002). 200
Our analysis of the Hirudo EST libraries led to the identification of 4 other TLRs that are all 201
expressed in the nervous system but whose functions remain undescribed (Table 1). One of them, 202
namely HmTLR3 was recently further analyzed and appeared to be up-regulated in the 203
bacterially challenged nerve cord (unpublished data). Interestingly, the LRR domain of HmTLR3 204
presents homologies with the vertebrates TLR8, known to be implicated both in the antiviral 205
response by recognizing single strand ARN and in the neuronal development (Ma et al., 2007). 206
The great homology of HmTLRs with vertebrate TLRs supports our interest to use the leech 207
model to understand the immune mechanisms developed by the CNS in mammals. PAMP 208
recognition by leech TLRs has yet to be elucidated to fully demonstrate the conservation 209
between the two systems. Indeed, it still has to be determined whether this is a direct recognition 210
as observed in mammals or an indirect recognition as described for the fruit fly (Leulier et al., 211
2008). 212
213
Nod-Like Receptors. The NLRs, consisting of more than 200 related family members, are 214
present in the cytosol and recognize intracellular MAMPs together with RLRs, TLRs, and C-type 215
lectin families (Motta et al., 2015). In addition to their response to intracellular pathogens, NLRs 216
have been shown to play important roles in distinct biological processes ranging from regulation 217
of antigen presentation, sensing metabolic changes in the cell, modulation of inflammation, 218
embryo development, cell death, and differentiation of the adaptive immune response (Lupfer 219
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and Kanneganti, 2013). While no NLR homologue has been found in the genomes of the 220
ecdysozoans Drosophila and Caenorhabditis elegans, a sequence related to NLR was found in 221
the CNS of the medicinal leech (Cuvillier-Hot et al., 2011). HmNLR shares best homologies 222
with NLRC3 receptors, considered as evolutionarily basal to the NLR family in vertebrates. 223
NLRC3 protein is an important cytosolic PRR that negatively regulates innate immune response 224
in mammals. In zebrafish, recent data demonstrate that NLRC3-like by preventing inappropriate 225
macrophage activation, contribute to normal microglial cell development (Microglia derived 226
from primitive macrophages that migrate into the brain during embryogenesis) (Shiau et al., 227
2013). Such role cannot be supported in the leech CNS since HmNLR has a brain tissue 228
expression restricted to neurons. Confocal microscopy data more precisely evidences an 229
accumulation of HmNLR in the submembranous compartment. This cytosolic distribution 230
reminds the localization of activated Nod2, whose membrane recruitment in human cells appears 231
as necessary for NF-kB activation post microbial challenge (Barnich et al., 2005). 232
The N-terminal tail of HmNLR (recognition domain) displays no conserved domain, nor does it 233
match with any known molecule in blast analysis. However, its orthologs detected in the genome 234
of Capitella, an annelid Polychaeta, do present a CARD domain upstream of the LRR domain. 235
Considering that the clitellates - among which the Hirudinae – probably derived from a 236
polychaete-like ancestor (Sperlin et al., 2009), it is possible that in the course of the evolution of 237
leeches the ligand-binding domain was conserved but not the upstream effector domains, 238
suggesting innovative transduction pathways. Interestingly, in the amphioxus genome also, some 239
sequences similar to vertebrate NLR without a NACHT domain were described (Huang et al., 240
2008), highlighting a NLR repertoire in non-vertebrates more complex than that of vertebrates. 241
The characterization of a NLR homologue in annelids reinforces the hypothesis of an ancient 242
origin for this family of cytosolic sentinels. 243
244
Viral sensing receptors. Multiple observations support the presence of viral sensing receptors in 245
the nerve cord of the leech. Several immune transcripts have been showed to be over-expressed 246
in the nerve cord after viral activation though polyI:C agents (Schikorski et al., 2008) (Fig. 2). 247
Silencing experiments performed to shut down HmTLR1 expression also resulted in increased 248
expression of the AMP Hm-lumbricin reflecting a siRNA sensing system in the leech nerve cord. 249
Among the PRRs that detect the intracellular presence of MAMPs, a virus sensing receptor RIG-250
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1 like DExD/H box RNA helicases (RLR) was detected in Hirudo EST (V. Cuvillier et al, 251
unpublished data). The presence of a great number of RNA helicases among the leech 252
transcripts, coupled with the presence of the RIG-1 orthologs, suggests the capacity for mounting 253
an efficient anti-viral immune response in Hirudo. Transcripts with strong homology to a number 254
of antiviral response factors, including Dicer, Drosha and Argonaute, were found in the Hirudo 255
database (Table 2) and we suspect the AMP lumbricin to possess anti-viral activities because of 256
its strong expression after viral challenges and its cytosolic localization. In leech, highly 257
conserved molecules related to serpins, eglin c, or leech-derived tryptase inhibitor (LDTI), which 258
in other systems are known to be active against viruses like HIV or Hepatitis C Virus NS3 259
protease (Auerswald et al., 1994; Martin et al., 1998), will need to be taken into account in 260
dissecting out the leech antiviral response. 261
262
Functions of PRRs in the response to neuronal injury and/or microbial infection of the leech 263
CNS: Under sterile conditions, genes encoding HmTLR1 or HmNLR appeared to be 264
differentially modulated during the regenerative process (Fig.2). Although the gene encoding 265
HmTLR1 was observed to be downregulated along with the CNS repair, the level of HmNLR 266
transcripts appeared to be up regulated a few days (3 days) post axotomy, suggesting a role of the 267
last one in the mid-term events engaged during neural regeneration (Cuvillier-Hot et al., 2011). 268
The regenerative process is known to be effective 7 days post injury of the leech CNS). 269
Interestingly, neuronal injury in mammals leads to the induction of NLRP1 and NLRP5, which 270
are believed to regulate caspase activation and apoptosis in injured neurons (Frederick Lo et al., 271
2008). This dual role of NLR family members sensing pathogens as well as damages fits 272
especially well to the neural context where immunity and tissue repair appear more and more as 273
intimately connected (Eming et al., 2009). Various hypotheses could explain the decrease of 274
HmTLR1 transcripts: (i) this receptor participates in limiting axonal growth as reported for 275
TLR3, (ii) HmTLR1 is not engaged in the regeneration of the injured CNS at all and/or (iii) 276
HmTLR1 is required for the regenerative process but because of the long lifespan of this protein 277
a neosynthesis is not needed. 278
The variation of the gene expression was also quantified in nerve cords experimentally infected 279
by various microbial derivatives (Fig. 2) (Cuvillier-Hot et al., 2011). Interestingly, a comparable 280
pattern of expression was obtained for both genes suggesting a communication between our two 281
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receptors upon microbial challenge of the injured leech nerve cords. Gram-positive bacteria and 282
Muramyl DiPeptide (MDP) appear as the best inducers of HmTLR1 and HmNLR genes. With 283
these conditions, our sensing receptors colocalized at the injured sites which, because of the solid 284
fibrous capsule surrounding the leech CNS, corresponds to the exclusive ways of 285
entrance/contact of/with microorganisms. These data also evoke a communication between 286
HmTLR1 and HmNLR in our model (Cuvillier-Hot et al., 2011). Chauhan and colleagues 287
showed that NOD2 synergizes TLR-induced inflammatory cytokine production in murine 288
microglia and astrocytes, illustrating the interplay that may exist between co-expressed TLR and 289
NLR receptors (Chauhan et al., 2009). Further investigations should be envisaged to study the 290
potential interplay between leech HmTLR1 and HmNLR in the CNS, both under homeostatic 291
conditions and following injury and / or infection. The role of Hm-TLR1 was elucidated 292
(Schikorski et al., 2009). Indeed, silencing of the HmTlr1 gene in the leech CNS demonstrated 293
that upon microbial challenge, this receptor is involved in the induction of the gene encoding the 294
chemokine Hmp43/EMAPII, an immune effector known to recruit phagocytic cells at the lesion 295
site. These data are reminiscent of some observations of rat microglial cells, which have been 296
reported to produce EMAPII after systemic injections of TLR agonists, such as polyinosine-297
polycytidylic acid (a TLR3 ligand) and R848 (a TLR 7/8 ligand) (Zhang and Schwarz, 2002). 298
The regulation of EMAPII by a TLR in both leech and mammals reinforces the great 299
conservation between these two models. Moreover, the existence of an immunity mediated by a 300
TLR in the leech CNS have showed for the first time an immune function of a TLR in a non-301
ecdysozoan model (i.e., in an invertebrate model that is different from C. elegans and D. 302
melanogaster) (Leulier and Lemaitre, 2008). We hypothesize that a co-evolutive process of the 303
sensor receptors might have took place between the parasite (i.e. the medicinal leech) and its 304
hosts (i.e. vertebrates including amphibians, fresh water fishes and mammals) presumably 305
resulting from their close contact with the same microorganisms. 306
307
2.2 PRR signalling pathways in the leech nervous system 308
309
In many species, including invertebrates and vertebrates, PRR activation triggers an intracellular 310
signalling pathway, followed by the regulation of defense genes. A survey of the medicinal leech 311
transcriptome and genome databases allowed revealing the presence in the leech of the main 312
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signaling molecules involved in the canonical vertebrate TLR pathways (Fig. 3 and table 1) 313
(Macagno et al., 2010). This stands in sharp contrast to other invertebrates, such as insects and 314
nematodes, for which the PRR pathways thus far appear to be less conserved, with many 315
components missing. Whether all the identified leech putative homologs indeed play similar 316
functional roles remains to be shown by further analysis, but their presence in the transcriptome 317
database adds once again support to the hypothesis that annelid genetic programs are more 318
closely related to those of vertebrates than are those of arthropods (Macagno et al., 2010) 319
(Gagniere et al., 2010). 320
At the level of the nervous system, data clearly showed that the leech nerve cord is able to 321
establish a specific neuroimmune response by discriminating microbial components after 322
microbial challenge and/or post injury. As detailed before, leech neural cells express various 323
PRRs, and in response produce immune specific effectors to fight encountered microbes and/or 324
promoting nerve repair (see below). Neurons and microglia express sensing receptors like Hm-325
TLR1, which is associated with chemokine production (i.e. EMAPII) in response to septic 326
challenge or lesion. To gain insights into the TLR signalling pathways involved in this 327
neuroimmune response, members of the Myeloid Differentiation factor 88 (MyD88) family were 328
investigated (Rodet et al., 2015). In mammals, it includes 5 adaptor proteins containing a TIR 329
domain, MyD88, MyD88-adapter-like (Mal), TIR-domain-containing adaptor protein-inducing 330
IFN beta (TRIF), TRIF-Related Adaptor Molecule (TRAM) and Sterile alpha and Armadillo-331
Motif-containing protein (SARM). All TLRs, except TLR3, recruit MyD88 to mediate innate 332
immune signalling. 333
In the leech nerve cord, two members of the MyD88 family: Hm-MyD88 and Hm-SARM have 334
been recently evidenced to be tightly regulated not only upon immune challenge but also during 335
CNS repair, suggesting their involvement in both processes (Rodet et al., 2015). Interestingly, a 336
stimulation of leech neurons with lipopolysaccharide (LPS) triggered a redistribution of Hm-337
MyD88 and Hm-TLR1 at the cell surface. To the best of our knowledge, these data showed for 338
the first time that differentiated neurons of the CNS could respond to LPS through a MyD88-339
dependent signalling pathway, while in mammals, studies describing the direct effect of LPS on 340
neurons and the outcomes of such treatment remain scarce and controversial. 341
342
3. I
MMUNE EFFECTORS PRODUCED BY NEURONS
343
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344
Once activated, sensing receptors of the leech CNS drive the production and release of numerous 345
molecular effectors like AMPs and cytokines. These factors promote the regenerative process 346
directly through their neurotrophic properties or indirectly thought the recruitment of immune 347
cells (microglia and blood cells) that accumulate and release neurotrophic factors at the lesion 348
site. 349
350
3.1. Neuronal antimicrobial substances with neurotrophic properties 351
352
The leech nervous system produces infection-inducible AMPs (Schikorski et al., 2008). Hm-353
lumbricin and neuromacin have been shown to be produced by microglial cells and by neurons 354
themselves in response to CNS injury. Microbial components differentially induce the 355
transcription, by microglial cells, of both antimicrobial peptide genes, the products of which 356
accumulate rapidly at sites in the CNS undergoing regeneration following axotomy. A 357
preparation of leech CNS depleted of microglial cells, allowed demonstrating the production of 358
AMPs by neurons themselves. 359
Neither neuromacin-like nor lumbricin-like molecules have been found in the genomes of 360
ecdysozoan invertebrates such as Caenorhabditis elegans and Drosophila melanogaster, 361
underlining the importance of enlarging the number of invertebrate models dedicated to study 362
innate immunity. 363
Surprisingly, in addition to manifesting antibacterial properties, neuromacin and Hm-lumbricin 364
exert impressive regenerative effects on the leech CNS. In vertebrates, one study provides 365
evidence for the positive effects of an antimicrobial peptide on the restoration of the functions of 366
a lesioned peripheric nerve. Indeed, the addition of neutrophil defensin NP-1 on the lesioned 367
sciatic nerve in rats leads to increase the rate of growth of regenerative nerve fibers by 30% 368
(Nozdrachev et al., 2006). These data observed in the leech were the first evidencing the 369
participation of AMPs produced by the nervous system itself in the regeneration process of the 370
CNS. However, multiple examples of neuropeptides, such as the alpha-Melanocyte Stimulating 371
Hormone in human and proenkephalin A-derived peptides in both mammals and/or leeches, have 372
also been described to possess antimicrobial properties in vitro (Tasiemski and Salzet, 2010). 373
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Thus, as in humans, antimicrobial peptides are involved in the innate immune system of the 374
leech CNS. 375
Neuromacin is a relative of theromacin, a cysteine-rich AMP first identified from the body fluid 376
of the leech Theromyzon tessulatum (Tasiemski et al., 2004) and later in Hirudo (Hm-377
theromacin) (Tasiemski and Salzet, 2010). Theromacin is synthesized by the large fat cells and 378
the blood cells while neuromacin is produced by the epithelial cells of the gut, the neurons and 379
the microglial cells of the leech CNS (Boidin-Wichlacz et al., 2012; Tasiemski et al., 2015; 380
Tasiemski et al., 2004). Theromacin possesses a longer C-terminal domain than neuromacin. 381
That results in two different conformations, resulting in different biological activities for the two 382
peptides (Jung et al., 2012). Further investigations revealed that neuromacin, like theromacin, 383
belongs to the macin family a new family of AMP within the superfamily of scorpion toxin-like 384
proteins. 385
The antimicrobial character of the macins is clearly demonstrated by their ability to permeabilize 386
membranes of B. megaterium within a few minutes, suggesting the bacterial membrane as target, 387
and its disruption as the mode of action (Jung et al., 2012). However, the macins showed 388
significant differences in their mechanistic behavior, pore-forming activity, activity not inhibited 389
by the presence of salt, were solely observed for neuromacin. This resistance to salt allows 390
neuromacin to exert its antimicrobial properties not only in the SNC but also in the gut lumen 391
where the osmolyte concentrations explode after each leech blood meal. We hypothesized that 392
neuromacin was probably selected instead of theromacin to cope with the variation in salt 393
concentrations in the gut environment. In this organ, neuromacin provides a protection against 394
invasive pathogenic bacteria and contribute to the unusual simplicity of the gut microflora of the 395
leech (Tasiemski et al., 2015). As this salt resistance was pH-dependent, it appears to be 396
mediated through the de-/protonation of the histidine residues that are missing in theromacin. 397
Moreover, neuromacin leads to aggregation of liposomes as well as Gram-negative bacteria 398
whereas theromacin does not. In fact, on the contrary of the surface properties of neuromacin, the 399
bipolar character of theromacin is insufficient for aggregation according to the barnacle model 400
i.e. dual electrostatic as well as hydrophobic peptide-membrane interaction applied in parallel to 401
two individual bacterial cells. Therefore, each bacterial cell can stick to several others, leading to 402
the formation of huge cell aggregates (Jung et al., 2012). 403
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Besides their antimicrobial activity, neuromacin and theromacin also exert a nerve-repair activity 404
(Jung et al., 2012). In Hirudo, the production sites of the leech macins are in accordance with 405
their regenerative effects on injured nerve cords. Indeed, theromacin that is produced by 406
circulating blood cells is released into the plasma that surrounds the nervous system whereas 407
neuromacin is directly produced by nerve cells and accumulates at the wounded site of the 408
central nervous system (Boidin-Wichlacz et al., 2012) (Schikorski et al., 2008). The leech plasma 409
enriched in AMPs, among them theromacin but also destabilase (a lysozyme like molecule), 410
stimulated the regeneration of the central nervous system suggesting a nerve-repair activity of 411
these antibiotic molecules. The neuronal repair process in leech does not include the de novo 412
regeneration of entire neurons (Duan et al., 2005). It is described so far as migration of microglia 413
to the site of lesion and an axon outgrowth which probably is cytoskeleton driven. Interestingly, 414
both neuromacin and theromacin not only induce nerve repair, i.e., the axonal regrowth in leech, 415
but they also increase the number of viable murine neuroblastoma cells. This observation might 416
extend the role of leech macins in nerve repair as they are able to modulate cytoskeletal functions 417
in leech and enhance the proliferation of neuroblastoma cells. Therefore, the nerve-repair process 418
in leech might also include the proliferation of neurons de novo. The mechanism of proliferation 419
induction remains to be determined. The fast uptake and initial inhomogeneous distribution of 420
theromacin might be a hint that endocytosis is involved. 421
The development of antimicrobial pharmaceuticals based on AMPs represents a promising 422
alternative approach in human anti-biotherapy. Beside their potential as templates for the 423
development of alternative antibiotics the AMPs’ various secondary” activities might be 424
beneficial for the development of pharmaceuticals suitable in other medical fields. In particular, 425
the nerve-repair activity of the leech macins or the proliferation effect exerted by theromacin, 426
neuromacin but also hydramacin are of particular interest (Jung et al., 2012). Further 427
investigations of the nerve-repair activity might eventually lead to findings that support the 428
development of pharmaceuticals effective against neural diseases, e.g., paraplegia, which is to 429
date purely speculative. Neuromacin was patented in this direction. 430
431
3.2. Neuronal cytokines with chemoattractant properties 432
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Among the effectors already discovered in leeches, HmTLR1 is linked to the cytokine related to 434
EMAP II in the context on the brain immune response after crush (Schikorski et al., 2009) (Table 435
3). HmEMAP II is the first cytokine-related molecule characterized in nervous system 436
invertebrates. The cytokine EMAPII has been suggested to be a marker of microglial cell 437
reactivity in the mammalian CNS (Schluesener et al., 1999; Schluesener et al., 1997; Tas and 438
Murray, 1996). Activated microglia of injured brain tissue ensuing from inflammation or 439
neurodegeneration have been shown to produce high levels of EMAPII (Mueller et al., 2003). 440
This cytokine was initially isolated from cultures of methylcholanthrene A-induced fibrosarcoma 441
cells and constitutes the mature product of the C-terminal region of a 43 kDa precursor referred 442
as the multi-synthetase complex p43, a component of the aminoacyl-tRNA synthetase complex 443
involved in protein synthesis in mammals (Shalak et al., 2001). Numerous studies have described 444
the pleiotropic biological activities of EMAPII and its precursor (van Horssen et al., 2006). At 445
the peripheral level, in vitro studies have demonstrated that EMAPII: (i) participates in the 446
recruitment of polymorphonuclear leukocytes and mononuclear phagocytes, (ii) promotes 447
endothelial apoptosis, (iii) enhances the expression of some other cytokines, and (iv) stimulates 448
dermal proliferation, wound repair, and graft neovascularization (Mueller et al., 2003). 449
Interestingly, even if EMAPII is now considered as a modulator of inflammatory reactions 450
within the peripheral innate immune response, its exact biological function in the neural immune 451
response of the CNS has yet to be elucidated. 452
The detection of Hmp43/EMAPII in the microglial cells accumulated at the injured site of the 453
leech CNS underlines some similarities of the inflammatory response of the nerve cord in our 454
model with that of the human brain (Mueller et al., 2003). In the leech, however, neuronal cells 455
also contribute to the production of HmEMAPII, as demonstrated in the preparation of leech 456
CNS depleted of microglial cells. By favoring the recruitment of microglial cells to the 457
axotomized site, EMAPII indirectly contributes to neural repair and to the antimicrobial response 458
of the leech CNS. Indeed, recruited microglial cells have been described to participate in the 459
phagocytosis of damaged tissues and in the regeneration process by producing laminin, an 460
extracellular matrix molecule known to promote neurite outgrowth and antimicrobial peptides, 461
which exert neurotrophic activities (Schikorski et al., 2008; von Bernhardi and Muller et al. , 462
1995). 463
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Besides EMAP, other cytokines are produced by the leech nervous system e.g. the one related to 464
human interleukin-16 (IL-16). Hm-IL-16 protein present in the neurons, is rapidly transported 465
and stored along the axonal processes to promote the recruitment of microglial cells to the 466
injured axons. The ability of Hm-IL-16 to recruit microglial cells to sites of CNS injury suggests 467
a role for Hm-IL-16 in the crosstalk between neurons and microglia in the leech CNS repair. 468
Interestingly, in addition to its chemo attractive property, HmIL-16 is able to promote human 469
CD4+ T cells migration thus showing functional analogies of Hirudo IL-16 (HmIL-16) with 470
human IL-16 (Croq et al., 2009). 471
Other molecules (granulin, SOCS, TNF alpha…..) already known for their cytokinic activity in 472
the nervous system of mammals have been detected in the leech CNS. Further investigations are 473
needed to determine their neuroimmune functions in Hirudo (Table 3). 474
475
4. C
ONCLUSION AND FUTURE DIRECTIONS
476
477
As in mammals, the leech nervous system uses a common panel of proteins to initiate 478
antiinfectious responses and regrowth programs. The relative simplicity of the leech CNS in 479
combination with its having complex mechanisms to react to infection suggests that the study of 480
the neural immunity in H. medicinalis will contribute to a better understanding of the implication 481
of immune molecules in the neural repair of the CNS in mammals. Interestingly a significant and 482
antigen-specific increase of the level of AMPs was recently detected in the nerve cords dissected 483
from leeches exposed to different alive bacteria added to their water environment (Fig. 4). This 484
differential gene expression of the leech AMPs observed in the CNS might result from a 485
peripheral signal induced by the bacteria in contact with the leech skin as observed for C. 486
elegans but also from bacteria that enter the gut and interact with the leech immunity and gut 487
microflora (Kawli et al., 2010). Further investigations in this direction are planned to clearly 488
establish whether there is a link between epidermal and neural immunity or/and between the gut 489
microbial community and the neural immunity of the leech. The fact that the medicinal leech 490
constitutes a model system of gut symbiosis reinforces the interest to explore this field (Bomar 491
and Graf, 2012; Nelson and Graf, 2012; Nyholm and Graf, 2012; Ott et al., 2014). An effect of 492
the microbiome that is not restricted to the digestive tract might be assumed taking into account 493
the growing evidence of the multiple roles that exert gut symbiotic bacteria on their host 494
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physiology and adaptation (Maranduba et al., 2015; Ray et al., 2015). Moreover it would be also 495
interesting to determine whether Hirudo uses its nervous system to respond to diverse microbial 496
cues, and engages it in a protective behavioral avoidance response to environmental pathogens 497
and/or a selective behavior of the environmental bacteria composing its gut microbiota. Indeed, 498
this behavior also constitutes an immune response, with sensors and recognition systems that 499
drives a protective response following a learning experience. 500
501
Acknowledgments 502
This work was supported by the University of Lille, the CNRS and the NSF. We would like to 503
sincerely acknowledge D. Schikorski, C. Boidin-Wichlacz, V. Cuvillier, F. Rodet, S. Jung, J. 504
Grotzinger and M. Leippe and for their contribution in this research project. 505
506
507
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Prehaud, C., Megret, F., Lafage, M., Lafon, M., 2005. Virus infection switches TLR-3-positive 626 human neurons to become strong producers of beta interferon. J Virol 79, 12893-12904. 627 Pujol, N., Link, E.M., Liu, L.X., Kurz, C.L., Alloing, G., Tan, M.W., Ray, K.P., Solari, R., 628 Johnson, C.D., Ewbank, J.J., 2001. A reverse genetic analysis of components of the Toll 629 signaling pathway in Caenorhabditis elegans. Curr Biol 11, 809-821. 630 Rauta, P.R., Samanta, M., Dash, H.R., Nayak, B., Das, S., 2014. Toll-like receptors (TLRs) in 631 aquatic animals: signaling pathways, expressions and immune responses. Immunol Lett 158, 14-632 24. 633 Ray, K., 2015. Gut microbiota: Host-microbe interactions and the enteric nervous system: a new 634 connection? Nat Rev Gastroenterol Hepatol 12, 311. 635 Rodet, F., Tasiemski, A., Boidin-Wichlacz, C., Van Camp, C., Vuillaume, C., Slomianny, C., 636 Salzet, M., 2015. Hm-MyD88 and Hm-SARM: two key regulators of the neuroimmune system 637 and neural repair in the medicinal leech. Sci Rep 5, 9624. 638 Russo, M.V., McGavern, D.B., 2015. Immune Surveillance of the CNS following Infection and 639 Injury. Trends Immunol 36, 637-650. 640 Schikorski, D., Cuvillier-Hot, V., Boidin-Wichlacz, C., Slomianny, C., Salzet, M., Tasiemski, 641 A., 2009. Deciphering the immune function and regulation by a TLR of the cytokine EMAPII in 642 the lesioned central nervous system using a leech model. J Immunol 183, 7119-7128. 643 Schikorski, D., Cuvillier-Hot, V., Leippe, M., Boidin-Wichlacz, C., Slomianny, C., Macagno, E., 644 Salzet, M., Tasiemski, A., 2008. Microbial challenge promotes the regenerative process of the 645 injured central nervous system of the medicinal leech by inducing the synthesis of antimicrobial 646 peptides in neurons and microglia. J Immunol 181, 1083-1095. 647 Schluesener, H.J., Seid, K., Meyermann, R., 1999. Effects of autoantigen and dexamethasone 648 treatment on expression of endothelial-monocyte activating polypeptide II and allograft-649 inflammatory factor-1 by activated macrophages and microglial cells in lesions of experimental 650 autoimmune encephalomyelitis, neuritis and uveitis. Acta Neuropathol (Berl) 97, 119-126. 651 Schluesener, H.J., Seid, K., Zhao, Y., Meyermann, R., 1997. Localization of endothelial-652 monocyte-activating polypeptide II (EMAP II), a novel proinflammatory cytokine, to lesions of 653 experimental autoimmune encephalomyelitis, neuritis and uveitis: expression by monocytes and 654 activated microglial cells. Glia 20, 365-372. 655 Shalak, V., Kaminska, M., Mitnacht-Kraus, R., Vandenabeele, P., Clauss, M., Mirande, M., 656 2001. The EMAPII cytokine is released from the mammalian multisynthetase complex after 657 cleavage of its p43/proEMAPII component. J Biol Chem 276, 23769-23776. 658 Shiau, C.E., Monk, K.R., Joo, W., Talbot, W.S., 2013. An anti-inflammatory NOD-like receptor 659 is required for microglia development. Cell Rep 5, 1342-1352. 660 Smith, P.J., Howes, E.A., Treherne, J.E., 1987. Mechanisms of glial regeneration in an insect 661 central nervous system. J Exp Biol 132, 59-78. 662 Steiner, H., Hultmark, D., Engstrom, A., Bennich, H., Boman, H.G., 1981. Sequence and 663 specificity of two antibacterial proteins involved in insect immunity. Nature 292, 246-248. 664 Tas, M.P., Murray, J.C., 1996. Endothelial-monocyte-activating polypeptide II. Int J Biochem 665 Cell Biol 28, 837-841. 666 Tasiemski, A., Massol, F., Cuvillier-Hot, V., Boidin-Wichlacz, C., Roger, E., Rodet, F., 667 Fournier, I., Thomas, F., Salzet, M., 2015. Reciprocal immune benefit based on complementary 668 production of antibiotics by the leech Hirudo verbana and its gut symbiont Aeromonas veronii. 669 Sci Rep 5, 17498. 670
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Tasiemski, A., Salzet, M., 2010. Leech immunity: from brain to peripheral responses. Adv Exp 671 Med Biol 708, 80-104. 672 Tasiemski, A., Vandenbulcke, F., Mitta, G., Lemoine, J., Lefebvre, C., Sautiere, P.E., Salzet, M., 673 2004. Molecular characterization of two novel antibacterial peptides inducible upon bacterial 674 challenge in an annelid, the leech Theromyzon tessulatum. J Biol Chem 279, 30973-30982. 675 Tauszig, S., Jouanguy, E., Hoffmann, J.A., Imler, J.L., 2000. Toll-related receptors and the 676 control of antimicrobial peptide expression in Drosophila. Proc Natl Acad Sci U S A 97, 10520-677 10525. 678 van Horssen, R., Eggermont, A.M., ten Hagen, T.L., 2006. Endothelial monocyte-activating 679 polypeptide-II and its functions in (patho)physiological processes. Cytokine Growth Factor Rev 680 17, 339-348. 681 von Bernhardi, R., Muller, K.J., 1995. Repair of the central nervous system: lessons from lesions 682 in leeches. J Neurobiol 27, 353-366. 683 Wharton, K.A., Jr., Crews, S.T., 1993. CNS midline enhancers of the Drosophila slit and Toll 684 genes. Mech Dev 40, 141-154. 685 Wrona, D., 2006. Neural-immune interactions: an integrative view of the bidirectional 686 relationship between the brain and immune systems. J Neuroimmunol 172, 38-58. 687 Zhang, F.R., Schwarz, M.A., 2002. Pro-EMAP II is not primarily cleaved by caspase-3 and -7. 688 Am J Physiol Lung Cell Mol Physiol 282, L1239-1244. 689
690
691
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Figure legends 692
693
Figure 1: (a) Schematic cross section of H. medicinalisbody. The CNS is enclosed within the 694
ventral blood sinus. (b) Defined amount of bacteria (mix of heat-killed M. nishino: Micrococcus 695
nishinomiyaensis and A. hydrophila: Aeromonas hydrophila at 3x10
7
CFU/ml) promotes the 696
regeneration process relative to sterile conditions (Schikorski, 2008). The Gram-positive and 697
Gram-negative bacteria M. nishino and A. hydrophila, respectively, were isolated from the 698
natural environment of Hirudo. Sectioning of one side of the paired connective nerve linking 699
adjacent segmental ganglia was performed on excised nerve cords maintained in culture. To 700
monitor the progress of nerve repair, micrographs of the damaged nerve cords were taken every 701
24 h, in the presence or absence of bacteria. Under sterile conditions, as documented in the left 702
column, restoration of the connective nerve across the cut begins at ~4 days post-axotomy (panel 703
J 4) and is finished 4 days later. In comparison, nerve repair is evident sooner in the presence of 704
a controlled number of bacteria, reconnection starting after 2 days or 3 days (right column). 705
706
Figure 2: Modulation of the gene expression of leech PRRs and immune effectors in nerve cords 707
incubated for 6 hours with various microbial components (2 µg/ml of LTA: lipoteichoïc acid; 10 708
µg/ml of MDP: muramyl dipeptide; 100 ng/ml of LPS: lipopolysaccharides), heat killed bacteria 709
at a concentration favoring the regenerative process (see Fig. 1b); VSV: Vesicular stomatitis 710
virus or viral mimetic (10 µg/ml of polyI:C). Incubations without microbial components or 711
bacteria were performed in the same conditions as controls. For the details see: Cuvillier-Hot et 712
al., 2011; Schikorski et al.,2008; Schikorski et al., 2009. 713
714
Figure 3: TLR pathways in the leech CNS. Analysis of the Hirudo transcriptome database 715
reveals the presence of putative homologs of nearly all factors reported to play critical roles in 716
human TLR pathways. Framed E values give the homologies of the leech proteins with their 717
counterparts identified in Ce : Caenorhabditis elegans, Dm : Drosophila melanogaster and Hs : 718
Homo sapiens. Some of them (E values 0) has already been entirely characterized. 719
720
Figure 4: The level of AMP expression is enhanced in the CNS of leeches that have swum for 6 721
hours in water heavily enriched in a mix of alive bacteria (A. nishinomyaensis and A. hydrophila 722
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10
7
CFU per ml) in comparison with leeches that have swum in water not enriched in bacteria. 723
Adult leeches were maintained at room temperature in sterile artificial pond water for one week 724
before starting the experiment (i.e. addition or not of bacteria to the water tank). Dissection of 725
the nerve cords, RT-qPCR and statistical analysis were performed according to the methods 726
described in Schikorski et al., 2008. 727
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Table 1: Repertoire of PRRs and elements of the PRR signalling pathways expressed in the
leech CNS.
E
-
4
Toll
-
Like Receptors (TLRs)
2e
-
17 to 2e
-
19
Hm
Nod Like Receptor (NLR)
0
Hm
RIG
-
1 like Receptors (RLRs)
0
Immunoglobulin superfamily
receptors
6.4e
-
16 to 1.5e
-
118
Hm
-
Myeloid differentiation factor 88 (MyD88)
0
Evolutionarily conserved signalling intermediate in toll pathways
(ECSIT) 8.1e-20
Hm
-
Sterile alpha and TIR motif
-
containing protein (SARM)
0
Toll
-
interacting protein (TOLLIP)
1.1e
-
32
Interleukin
-
1 receptor
-
associated kinase 4 (IRAK
-
4)
3.7e
-
34
Tumor necrosis factor (TNF) receptor
-
associated factor 3 (TRAF
-
3)
1.3e
-
52
TNF receptor associated factor 6 (TRAF
-
6)
1.7e
-
21
P38 mitogen activated protein kinase (MAPK)
6.3e
-
95
NF
-
kappa
-
B p105 subunit (Contains: NFkB p50 subunit)
4.5e
-
29
IKK
-
like protein
4.4e
-
45
Interferon Regulatory Factor (IRF)
1.3e
-
16 to 2.2e
-
17
similar to interferon gamma
-
inducible protein 30
8e
-
32
Fas Associated
via
Death Domain (FADD)
2.3e
-
18
Caspases
3/7
3.8e
-
53 to 2.7e
-
59
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Table 2: Antimicrobial response elements produced by the leech CNS.
E-values
Hm
Lumbricin (AMP)
0
Hm
Theromyzin (AMP)
0
Hm
Neuromacin (AMP)
0
Destabilase 1 (lysozyme
-
like)
0
Eglin
-
C
2.3e
-
36
LPS Binding Protein/
Bactericidal Permeability Increasing protein
(LBP/BPI) 6.2e-40
Dicer
7.3 e
-
91
Drosha
6.2e
-
76
Argonaute
-
like protein
1.9e
-
98
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Table 3: (a) Cytokine related molecules synthesized by the leech CNS (b) Level of expression in
the CNS incubated with a controlled number of bacteria promoting the regenerative process.
E values
Hm p43/Endothelial Monocyte-Activating Polypeptide 2 (EMAP2) 0
TNF alpha 6.6e-23
Granulins 1.8e-34
SOCS 6.9e 27
Hm Interleukin-16 0
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Figure 1
Blood
Dorsal Sinus
Intestine
Diverticulum
Lateral
Sinus
Ventral Sinus
CNS
Segmental
Ganglion
Epidermis
Sterile conditions Bacterial infection
B
A
Day(s) post- axotomy
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2
4
6
8
10
12
14
16
18
20
Hmlumbricin
Neuromacin
HmEMAPII
HmNLR
HmTLR1
HmRLR
****
** **** **
**
**
***
Gram + Gram - viruses
Level of gene expression
Figure 2
**
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Figure 3
LEECH HUMAN
Direct
recognition of
PAMPs by TLRs
Direct or indirect
recognition of
PAMPs by TLRs ?
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Figure 4
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
T 0h T 6h Sterile
water
T 6h Water
enriched in
bacteria
Lumbricin 1.00 2.78 5.50
Neuromacin 1.00 0.98 2.30
Lumbricin
Neuromacin
Level of gene expression
**
**
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The medicinal leech is an excellent model system for exploring fundamental questions about the
interaction of the nervous and innate immune systems
As in mammals, the leech CNS uses a common panel of proteins to initiate antiinfectious responses
and regenerative programs
Leech neurons produce antimicrobial peptides having neurotrophic activities
Leech neurons express microbial sensing receptors
  • ... Another important criticism comes from the obvious notion that cognition, behavioral complexity and neuroimmune interconnection are far from being the preserve of species endowed with an adaptive immune system. Invertebrates such as for instance the medicinal leech, exhibit finely-tuned neuroimmune interactions ( Tasiemski and Salzet, 2017) and proceed to decision-makings when executing complex locomotor behaviors ( Esch et al., 2002;Harley et al., 2013). To answer this valid criticism, one may argue that the dichotomy established between innate and adaptive immunity is possibly specious and that "custom-fit immunity" ( Rimer et al., 2014) i.e., basically, adaptive immunity, operate in all forms of life ( Rimer et al., 2014). ...
    ... I propose the term of neuroimmune autoperception to depict the whole of such immune mechanisms. As previously put forward by Jean Dausset, the discoverer of the MHC system, adaptive immunity could be thus "regarded as a late evolution from a self-recognition system" ( Tasiemski and Salzet, 2017). In this extended view of autoimmunity, the neuroimmune co-development/co-evolution model might be relevant in a large range of species. ...
    Article
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    In the last decades, increasingly robust experimental approaches have formally demonstrated that autoimmunity is a physiological process involved in a large range of functions including cognition. On this basis, the recently enunciated “brain superautoantigens” theory proposes that autoimmunity has been a driving force of cognitive evolution. It is notably suggested that the immune and nervous systems have somehow co-evolved and exerted a mutual selection pressure benefiting to both systems. In this two-way process, the evolutionary-determined emergence of neurons expressing specific immunogenic antigens (brain superautoantigens) has exerted a selection pressure on immune genes shaping the T-cell repertoire. Such a selection pressure on immune genes has translated into the emergence of a finely tuned autoimmune T-cell repertoire that promotes cognition. In another hand, the evolutionary-determined emergence of brain-autoreactive T-cells has exerted a selection pressure on neural genes coding for brain superautoantigens. Such a selection pressure has translated into the emergence of a neural repertoire (defined here as the whole of neurons, synapses and non-neuronal cells involved in cognitive functions) expressing brain superautoantigens. Overall, the brain superautoantigens theory suggests that cognitive evolution might have been primarily driven by internal cues rather than external environmental conditions. Importantly, while providing a unique molecular connection between neural and T-cell repertoires under physiological conditions, brain superautoantigens may also constitute an Achilles heel responsible for the particular susceptibility of Homo sapiens to “neuroimmune co-pathologies” i.e., disorders affecting both neural and T-cell repertoires. These may notably include paraneoplastic syndromes, multiple sclerosis as well as autism, schizophrenia and neurodegenerative diseases. In the context of this theoretical frame, a specific emphasis is given here to the potential evolutionary role exerted by two families of genes, namely the MHC class II genes, involved in antigen presentation to T-cells, and the Foxp genes, which play crucial roles in language (Foxp2) and the regulation of autoimmunity (Foxp3).
  • ... They change their morphology from ramified to amoeboid shape upon activation, thus facilitating their recruitment towards lesioned areas [7]. Microglia are the only migrating cells to be recruited at the injury site within 24 h post-lesion [9,10] and promote a regenerative process [11,12]. Indeed, if microglia accumulation is delayed at lesions, the axonal sprouting is consequently affected [13]. ...
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    In healthy or pathological brains, the neuroinflammatory state is supported by a strong communication involving microglia and neurons. Recent studies indicate that extracellular vesicles (EVs), including exosomes and microvesicles, play a key role in the physiological interactions between cells allowing central nervous system (CNS) development and/or integrity. The present report used medicinal leech CNS to investigate microglia/neuron crosstalk from ex vivo approaches as well as primary cultures. The results demonstrated a large production of exosomes from microglia. Their incubation to primary neuronal cultures showed a strong interaction with neurites. In addition, neurite outgrowth assays demonstrated microglia exosomes to exhibit significant neurotrophic activities using at least a Transforming Growth Factor beta (TGF-β) family member, called nGDF (nervous Growth/Differentiation Factor). Of interest, the results also showed an EV-mediated dialog between leech microglia and rat cells highlighting this communication to be more a matter of molecules than of species. Taken together, the present report brings a new insight into the microglia/neuron crosstalk in CNS and would help deciphering the molecular evolution of such a cell communication in brain.
  • ... Strikingly, both assays strongly suggested that RNASET2 affects bacterial integrity. Although several antimicrobial peptides produced by medicinal leech have been described [53,54], to our knowledge, this is the first report of the occurrence of an antibacterial activity of a ribonuclease protein in this animal model. The expres- sion of RNASET2 seemed not to interfere with bacterial viability as outlined from viability assay in co-cultures. ...
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    Recent studies demonstrated that allograft inflammatory factor-1 (AIF-1) and RNASET2 act as chemoattractants for macrophages and modulate the inflammatory processes in both vertebrates and invertebrates. The expression of these proteins significantly increases after bacterial infection; however, the mechanisms by which they regulate the innate immune response are still poorly defined. Here, we evaluate the effect of bacterial lipopolysaccharide injection on the expression pattern of these genes and the interrelation between them during innate immune response in the medicinal leech, an invertebrate model with a simple anatomy and a marked similarity with vertebrates in inflammatory processes. Collectively, prokaryotic-eukaryotic co-cultures and in vivo infection assays suggest that RNASET2 and AIF-1 play a crucial role in orchestrating a functional cross-talk between granulocytes and macrophages in leeches, resulting in the activation of an effective response against pathogen infection. RNASET2, firstly released by granulocytes, likely plays an early antibacterial role. Subsequently, AIF-1+ RNASET2-recruited macrophages further recruit other macrophages to potentiate the antibacterial inflammatory response. These experimental data are in keeping with the notion of RNA-SET2 acting as an alarmin-like molecule whose role is to locally transmit a "danger" signal (such as a bacterial infection) to the innate immune system in order to trigger an appropriate host response.
  • ... For what concerns Mollusca, early cellular studies ( Canesi et al., 2002;MatriconGondran and Letocart, 1999;Takahashi and Muroga, 2008) have been followed by extensive molecular surveys in gastropods ( Adema et al., 2017;Coustau et al., 2015;Pila et al., 2017), cepha- lopods ( Castillo et al., 2015;Gestal and Castellanos-Martínez, 2015) and bivalves Song et al., 2015;Zhang et al., 2015). A minor interest has been directed to Annelida, with studies targeting immune cells (Boidin-Wichlacz et al., 2011;Vetvicka and Sima, 2009) and immune genes (Altincicek and Vilcinskas, 2007;Nyholm et al., 2012;Tasiemski and Salzet, 2017) of polychaetes, oligochaetes and hirudinean worms. ...
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    Microglia are phagocytic cells that form the basis of the brain's immune system. They derive from primitive macrophages that migrate into the brain during embryogenesis, but the genetic control of microglial development remains elusive. Starting with a genetic screen in zebrafish, we show that the noncanonical NOD-like receptor (NLR) nlrc3-like is essential for microglial formation. Although most NLRs trigger inflammatory signaling, nlrc3-like acts cell autonomously in microglia precursor cells to suppress unwarranted inflammation in the absence of overt immune challenge. In nlrc3-like mutants, primitive macrophages initiate a systemic inflammatory response with increased proinflammatory cytokines and actively aggregate instead of migrating into the brain to form microglia. NLRC3-like requires both its pyrin and NACHT domains, and it can bind the inflammasome component apoptosis-associated speck-like protein. Our studies suggest that NLRC3-like may regulate the inflammasome and other inflammatory pathways. Together, these results demonstrate that NLRC3-like prevents inappropriate macrophage activation, thereby allowing normal microglial development.
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    The innate system's recognition of non-self and danger signals is mediated by a limited number of germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPs). Toll-like receptors (TLRs) are single, non-catalytic, membrane-spanning PRRs present in invertebrates and vertebrates. They act by specifically recognizing PAMPs of a variety of microbes and activate signaling cascades to induce innate immunity. A large number of TLRs have been identified in various aquatic animals of phyla Cnidaria, Annelida, Mollusca, Arthropoda, Echinodermata and Chordata. TLRs of aquatic and warm-blooded higher animals exhibit some distinctive features due to their diverse evolutionary lineages. However, majority of them share conserve signaling pathways in pathogen recognition and innate immunity. Functional analysis of novel TLRs in aquatic animals are very important in understanding the comparative immunology between warm-blooded and aquatic animals. In additions to innate immunity, recent reports have highlighted the additional roles of TLRs in adaptive immunity. Therefore, vaccines against many critical diseases of aquatic animals may be made more effective by supplementing TLR activators which will stimulate dendritic cells. This article describes updated information of TLRs in aquatic animals and their structural and functional relationship with warm-blooded animals.