Microglial Implication in Modulation of Inflammation in leech CNS – MIMIC

The first approach is based on the easy collection of the leech CNS. Collected without the blood system (impossible to realize in mammals) it is incubated in vitro in order to do experimental lesions. The analyses of molecules that are produced during the respons to injury are performed at the gene and protein levels.
Then the recruitment of microglial cells is studied inside the injured tissues (in vivo and ex vivo) thanks to the use of specific antibodies against the molecules of interest or against markers of microglia. The in situ observation is enriched by in vitro biological assays based on cell mobility towards a gradient of chemotactic factor.
Finally the understanding of interactions between target cells and chemoattractants is analyzed by characterizing the receptors for each molecule on microglial cells. That requires the purification of those receptors on a chromatography column by immune-affinity. This approach is still developed by using protein extracts from whole CNS or from specific cell populations. The response is actually based on a few numbers of cells which are exposing receptors for chemoattractants. Specific optimizations are thus necessary according to the specificities of each chemotactic factor for recruited microglial sub-populations.

This project allowed the characterization of several chemotactic molecules that are produced by neurons in order to attract microglial cells at the lesion site. That mobility is studied in a time-dependent manner and shows that microglial activation occurs in early step and is maintained 3 days following experimental injury. Presently two receptors for a chemotactic factor (HmC1q) were identified and allowed to better understand the mechanisms of movement for microglial sub-populations towards the lesion. After their identification, we have undertaken the large scale production of these chemotactic factors (recombinant forms) in order to develop in vitro studies. Indeed in addition to understand the process of microglial recruitment, the project would help to investigate the microglial functions of accumulated microglia. To do this, we initiated in vitro experiments allowing their activation by recombinant forms of chemotactic factors. The analyses of results are in progress. That will allow measuring the importance of microglial cells at lesions, for example by identifying secreted molecules, real microglia-related molecular signature communicating with cell environment in which damaged neurons are present. The valorization of results was triggered by two international publications, three international conferences and some collaborations with neurobiologists working on rat spinal cord injury or neurosurgeons.

Focused on a fundamental and basic science, the results we have got in the leech have to be specified and developed. But the achievement of knowledge on microglial involvement in the time-course of nerve repair allows having an better overview of neurodegenerative processes occurred in mammals. Thus leech molecules are ever used and tested in a model of rat spinal cord injury. Preliminary results show a significant and beneficial effect suggesting that leech molecules might activate some signalization mechanisms that are valuable to the nerve repair.

The results highlight the importance of HmC1q in microglia recruitment and were published in 2012 (1). Our advances in the understanding of microglia activation and the overview of leech convenient as tools in neurobiology were published in a review article in 2013 (2). In order to present the results and sensibilize the neurobiologists to the leech model, the studies were presented in international congresses in neurosciences and acta were published (3-5). The collaboration with neurobiologists using leech molecules in a model of rat spinal cord injury were valorized (3) and will be published very soon.
1. M. Tahtouh, A. Garçon-Bocquet, F. Croq, J. Vizioli, P.E. Sautière, C. Van Camp, M. Salzet, P. Nagnan-le Meillour, J. Pestel, C. Lefebvre. Interaction of HmC1q with leech microglial cells: involvement of C1qBP-related molecule in the induction of cell chemotaxis. Journal of Neuroinflammation 02/2012; 9:37.
2. F. Le Marrec-Croq, F. Drago, J. Vizioli, P.E. Sautière, and C. Lefebvre. The Leech Nervous System: A Valuable Model to Study the Microglia Involvement in Regenerative Processes. Clinical and Developmental Immunology 07/2013, in press ID 274019, dx.doi.org/10.1155/2013/274019.
3. I. Fournier, J. Franck, F. Croq, D. Cizkova, C. Lefebvre, J. Vizioli, P.E. Sautière, M. Salzet. Maldi imaging mass spectrometry: a novel technology for studying neurosciences. Glia 06/2013; 61(S1):S49-S216.
4. F. Drago, A. Accorsi, P.E. Sautière, F. Croq, C. Lefebvre, C. Van Camp, J. Vizioli. Characterization and modulation of HmIba1 as an activation marker for microglia in the invertebrate model, the leech Hirudo medicinalis. Glia 06/2013; 61(S1):S49-S216.
5. C. Lefebvre, A. Bocquet-Garçon, J. Vizioli, C. Van Camp, P.E. Sautière, F. Drago, M. Salzet, F. Croq. Microglia activation in the leech Hirudo medicinalis: HmC1q promotes the microglial accumulation through the distinct recognition of gC1qR and cC1qR receptors. Glia 06/2013; 61(S1):S49-S216.1.

Microglia are intrinsic components of the central nervous system (CNS). During pathological events in mammals, inflammatory processes implicate the resident microglia and the infiltration of peripheral immune cells of which macrophages. Because they are implicated as sensors of pathological changes in CNS tissue, the cytokines produced by microglial cells represent interesting markers of cell activation and contribute to reactive processes (Ransohoff et al., 2007). Functions of microglia appear to be complex as they exhibit both neuroprotective and neurotoxic effects. For a long time, some authors have focused on the understanding of mechanisms of microglial activation during neuropathological conditions in vivo and in vitro. But the limiting question is to discriminate the activated microglial cells from the peripheral infiltrating macrophages, a step which is a necessary prerequisite to understand successive steps occurring in CNS pathological situations (Prinz and Mildner, 2011).
In the present project, the medicinal leech Hirudo medicinalis is used to specify the activation processes of only resident microglial cells. In the laboratory for many years, the leech was studied due to its ability to naturally repair its central nervous system (CNS) following injury such as a crush or a complete section of the nerve cord (Coggeshall and Fawcett, 1964; Jansen and Nicholls, 1972; von Bernhardi and Muller, 1995). This experimental model offers original opportunities to study the molecular and cellular basis of the CNS repair processes leading to functional recovering. Of interest, when the leech CNS is injured, resident microglial cells migrate and accumulate at the site of lesion (Morgese et al., 1983). Importantly, this phenomenon is known to be essential for the usual sprouting of injured axons and does not require any infiltrating blood cells as we will describe in the proposal (Ngu et al., 2007).
The study will focus on (i) the complete characterization of leech chemotactic factors, already described as responsible of the microglial recruitment (HmIL-16 and HmC1q), (ii) the identification of nerve cells producing these molecules, (iii) the action kinetics of the different chemoattractants on microglia, (iv) and the study of different microglial subsets by identifying the specific receptors for the respective cytokines. This program aims to better understand the mechanisms of microglial recruitment in order to explore in further studies the crosstalk between neurons and the different microglia subsets, which is necessary to understand the balance of inflammation leading to the leech CNS repair.

References
Coggeshall RE, Fawcett DW (1964) The Fine Structure of the Central Nervous System of the Leech, Hirudo Medicinalis. J Neurophysiol 27:229-289.
Jansen JK, Nicholls JG (1972) Regeneration and changes in synaptic connections between individual nerve cells in the central nervous system of the leech. Proc Natl Acad Sci U S A 69:636-639.
Morgese VJ, Elliott EJ, Muller KJ (1983) Microglial movement to sites of nerve lesion in the leech CNS. Brain Res 272:166-170.
Ngu EM, Sahley CL, Muller KJ (2007) Reduced axon sprouting after treatment that diminishes microglia accumulation at lesions in the leech CNS. J Comp Neurol 503:101-109.
Prinz M, Mildner A (2011) Microglia in the CNS: Immigrants from another world. Glia 59:177-187.
Ransohoff RM, Liu L, Cardona AE (2007) Chemokines and chemokine receptors: multipurpose players in neuroinflammation. Int Rev Neurobiol 82:187-204.
von Bernhardi R, Muller KJ (1995) Repair of the central nervous system: lessons from lesions in leeches. J Neurobiol 27:353-366.