Clément Tavakoli

Elisa Cuccione

Chloe Dumot

David Cormode

Marlène Wiart

Hélène Elleaume

Emmanuel Brun

Chloé Dumot

Tracking cells in the brain of small animals using synchrotron multi-spectral phase contrast imaging

Keywords: X-ray spectral imaging, registration, brain imaging

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INTRODUCTION

Because it is fast and cost-effective, conventional attenuation-based X-ray Computed Tomography may be used as a noninvasive method for following up cellular therapies in small animal models [START_REF] Ashton | In vivo small animal micro-CT using nanoparticle contrast agents[END_REF]. Unfortunately, this technique faces some limitations in terms of resolution and contrast. Moreover, for separating the signal of contrast agents from similar endogenous signals, it often requires to image the animal before and after contrast agent injection, which may be a problem in terms of dose. Novel experimental CT techniques, namely Phase Contrast [START_REF] Bravin | X-ray phase-contrast imaging: from pre-clinical applications towards clinics[END_REF] and Spectral Imaging [START_REF] Thomlinson | K-edge subtraction synchrotron X-ray imaging in biomedical research[END_REF], that have been introduced in synchrotrons in the past years and recently translated to the clinics [START_REF] Willer | X-ray dark-field imaging of the human lung-A feasibility study on a deceased body[END_REF], [START_REF] Fehrenbach | Spectral CT and its specific values in the staging of patients with non-small cell lung cancer: technical possibilities and clinical impact[END_REF], may help to overcome those limitations. While they were developed in parallel, the aim of our study is to combine the two approaches for an improved segmentation of labelled-cells with a focus on small animal follow-up with a model of focal cerebral injury mimicking stroke infarct.

Stroke is the leading cause of long-term disability, resulting in an increasing need for a reliable and reproducible brain regeneration technique. Several types of regenerative therapies are currently being evaluated, the introduction of stem cells through intracerebral route proving to be associated with the best recovery to date compared to other administration routes. It is thus essential to develop imaging modalities that allow the monitoring of the administered cells in order to follow their fate in a non-invasive manner. Labelling the injected cells using gold nanoparticles (AuNPs) allows to monitor the cells with CT-based imaging techniques, in particular the novel approaches described above.

The data volume generated by such technique can be of hundreds of Gigabytes for one animal. Automatic, robust and rapid pipeline is therefore of paramount importance for large-scale studies. In this work, we have developed a pipeline devoted to the rigid segmentation of labelled cells. After a brief introduction of the physical phenomena we introduce the image processing techniques used in this study for the signal reconstruction method, we then describe the main features of our registration and segmentation approach.

SYNCHROTRON PHASE CONTRAST MULTI-SPECTRAL IMAGING

Physical principles

Material Decomposition, the task to separate X-ray images into two or more materials, is usually performed using the element specific energy-dependence of X-ray attenuation [START_REF] Alvarez | Energy-selective reconstructions in X-ray computerised tomography[END_REF], materialized by the linear attenuation coefficient µ of the element. This technique is currently being adopted in the clinics using either dual energy CT or Spectral Photon Counting CT scanners [START_REF] Mccollough | Dualand Multi-Energy CT: Principles, Technical Approaches, and Clinical Applications[END_REF]. Using a wave approach of light, X-ray Phase Contrast Imaging (PCI) is able not only to reconstruct the linear attenuation coefficient µ, but also the phase shift caused by the sample that can be quantified using the refractive index decrement  for a given element. Indeed, each element can be described by its refraction index 𝑛 = 1 -𝛿 + 𝑖𝛽 where  is the absorption index. Figure 1a shows in log scale the dependence in energy of the  and  indices for 3 elements/tissues. As one can see, like the linear attenuation coefficient, the absorption index shows a discontinuity at the K-edge of the element (80.7keV for gold). One can note that the / ratio can be up to 2180 for water at 40keV resulting in an increased contrast with phase sensitive imaging [START_REF] Bravin | X-ray phase-contrast imaging: from pre-clinical applications towards clinics[END_REF]. In the recent years, PCI has demonstrated, at multiple occasions, to increase the contrast in the images when compared to conventional attenuation-based CT imaging in several domains such as lungs [START_REF] Kitchen | CT dose reduction factors in the thousands using X-ray phase contrast[END_REF], breasts [START_REF] Castelli | Mammography with Synchrotron Radiation: First Clinical Experience with Phase-Detection Technique[END_REF] or joints [START_REF] Rougé-Labriet | X-ray phase contrast osteo-articular imaging: a pilot study on cadaveric human hands[END_REF]. Among all PCI experimental set-ups, here, we chose to use the Propagation Based Imaging [START_REF] Baruchel | X-ray optics and imaging with hard coherent synchrotron radiation[END_REF] technique which has the great advantage, over other PCI setups, of being simple and fast for in vivo applications. The experimental set-up is shown in figure 1b. The only difference with a conventional X-ray imaging set-up is the additional distance (z=11m) between the sample and the detector. PBI explores the phase shifts caused by variations in the refractive index and density of materials by capturing alterations in the measured intensities which can be observed through edge enhancement. From the raw signal that contains both absorption and refraction one can use phase retrieval algorithms. To limit the computation time, we used a GPU version of the so-called Paganin algorithm [START_REF] Paganin | Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object[END_REF]. This algorithm allows the retrieval of the linear attenuation coefficient (µ) assuming a unique / ratio over the sample. Although this assumption is completely wrong in the case of radiography of biological samples mixing low and high absorbing tissue, it surprisingly works well in CT mode due to the fact that locally this unique / ratio is true, as demonstrated in several studies [START_REF] Rougé-Labriet | X-ray phase contrast osteo-articular imaging: a pilot study on cadaveric human hands[END_REF], [START_REF] Tafforeau | Applications of X-ray Synchrotron microtomography for non-destructive 3D studies of paleontological specimens[END_REF].

The second imaging technique, also referred to K-edge imaging, uses the discontinuity of the  index around the K-edge of a specific element. In our study the cells were labelled with gold nanoparticles and CT images were acquired at two energies around the gold's K-edge. From these two acquisitions, after phase retrieval, we obtain 2 sets of data with linear coefficient µabove and µbelow that we can estimate as:

{ µ 𝑎𝑏𝑜𝑣𝑒 = µ 𝑀 1 ,𝐸 𝑎𝑏𝑜𝑣𝑒 . 𝑓 𝑀 1 + µ 𝑀 2 ,𝐸 𝑎𝑏𝑜𝑣𝑒 . 𝑓 𝑀 2 µ 𝑏𝑒𝑙𝑜𝑤 = µ 𝑀 1 ,𝐸 𝑏𝑒𝑙𝑜𝑤 . 𝑓 𝑀 1 + µ 𝑀 2 ,𝐸 𝑏𝑒𝑙𝑜𝑤 . 𝑓 𝑀 2 (1)
Where 𝑓 𝑀 1 and 𝑓 𝑀 2 are respectively the volume fraction of the material 𝑀 1 and 𝑀 2 of interest. µ 𝑀 1 ,𝐸 𝑎𝑏𝑜𝑣𝑒 is the linear coefficient for a given material at a given energy. So in our case, the concentration of gold can be retrieved by solving the linear system using the following formula:

𝑓 𝐴𝑢 = µ 𝑎𝑏𝑜𝑣𝑒 .µ 𝑊𝑎𝑡𝑒𝑟,𝐸 𝑏𝑒𝑙𝑜𝑤 -µ 𝑏𝑒𝑙𝑜𝑤 .µ 𝑊𝑎𝑡𝑒𝑟,𝐸 𝑎𝑏𝑜𝑣𝑒 µ 𝐻 2 𝑂,𝐸 𝑏𝑒𝑙𝑜𝑤 .µ 𝐴𝑢,𝐸 𝑎𝑏𝑜𝑣𝑒 -µ 𝐴𝑢,𝐸 𝑏𝑒𝑙𝑜𝑤 .µ 𝐻 2 𝑂,𝐸 𝑎𝑏𝑜𝑣𝑒 (2)

Material

We used the high intensity flux produced by the ESRF medical beamline (ID17) wiggler source which provides two quasicollimated monochromatic X-ray beams just below (79.7 keV) and above (81.7 keV) the K-edge of Gold (80.7 keV). A total of eleven rats with a model of focal cerebral injury were studied. They were administered with 500k AuNP-labelled cells and imaged at the synchrotron ten days post-transplantation. Once anesthetized, the animals were mounted on a scanning sample stage located at approximately 11 m from the detector. Sets of 2000 projections were collected for each 360-degree rotation. An optical system consisting of a scientific CMOS technology-based camera coupled to a Gd2O2S scintillator and an objective was used to record the X-ray images. The effective isotropic pixel size of the system was 21.3 µm. Due to the small height of the beam (4 mm), multiple CT acquisitions were necessary to image the complete region of interests (maximum 17 mm in height). A total time of 12 minutes was necessary to collect the whole tomographic data set for one rat for the 2 data volumes. After phase retrieval a standard filtered back projection was used for the CT reconstructions, as described in [START_REF] Mirone | The PyHST2 hybrid distributed code for high speed tomographic reconstruction with iterative reconstruction and a priori knowledge capabilities[END_REF].

IMAGE ANALYSIS PIPELINE

As the images were acquired in vivo sequentially, the rat movements had to be considered. Figure 2 presents the entire pipeline developed for the segmentation of AuNP-labelled cells. We chose to register the images based on the skull of the rats. This allowed us to select portions of the 3D data sets that are of interest, thereby reducing the size of the volumes to study and accelerate the speed of the registration algorithm. Finally, once the volumes have been registered and subtracted, the gold concentrations could be measured, corresponding to the AuNP-labelled cells injected into the brain. The details of each process of the pipeline is described in the following sub-sections.

Figure 2. The 4 steps of the cells tracking pipeline resulting in a map of gold concentrations obtained from the two volumes acquired above and below the gold K-edge.

Skull segmentation

The phase contrast method allows us to easily identify the intensity values corresponding to the structures of interest in our images, in this context: the skull of the rat. In fact, as seen on Figure 3, an analysis of the volume histogram allows the distinction between three main intensity groups: the first one corresponding to the air, the second one to the soft tissues and the plastic supporting the rat, the last one to the bones and the gold in the injected cells.

An initial step consists in performing a threshold operation so that only the bone remains in the image. By performing a simple gradient analysis on the histogram, we can easily identify the peaks specific to the 3 parts described and thus calculate the optimal bone threshold value.

Once the bones have been segmented, we need to extract only the cranial skull and get rid of the other segmented structures such as jaws, labelled cells and teeth. To do so we labelled the different structures by a connected component analysis. For

Skull Segmentation

Registration

Gold Extraction Subtraction

Above Volume Below Volume

this purpose, we use the IPSDK library (Reactiv'IP, Grenoble France), which, as opposed to traditional libraries, has the great advantage of being adapted to large volumes, thus allowing us to gain a significant amount of time using three dimensional operations. We thereby end up with a list of labeled regions with characteristics associated to each of them, the size of which turns out to be an ideal discriminating factor for the identification of the cranial skull. Indeed, its size is at least ten times larger than the smaller structures (particularly the jaws). This method has proven to be robust for all the volumes studied at the different energies.

Registration

As explained previously, our intention consists in comparing two volumes 𝑉 𝐵𝑒𝑙𝑜𝑤 and 𝑉 𝐴𝑏𝑜𝑣𝑒 of the same sample acquired at two different energy levels (E Below =79.7 keV and E Above =81.7 keV). As can be seen on Figure 4, the variation in the position of the rat on the two volumes is too great to be neglected. The subfigure 4c shows indeed the subtraction of the 2 raw images (4a and 4b). It is therefore necessary to match the voxels representing the same point in space from one volume to the other and do the so-called registration of the two volumes.

Registration is a complex task with several parameters including the optimizer, the transformation, the interpolator and finally the similarity metric. If the transformation is trivially rigid in our case, finding the other parameters is not straightforward. We assume that, in our case, the volumes were acquired using the same modality and only differ by a basic geometric translation and rotation. We empirically chose to split this registration into two steps: a first transformation 𝑇 1 consisting of a simple and fast 3D translation closing most of the gap between the two volumes, followed by a second transformation 𝑇 2 applying a rotation and translation to the space given 3 Euler angles and a 3D translation. We chose to compare two typical intensity-based metrics such as the mean squared intensity difference (MSQ) and crosscorrelation (CC) in terms of rapidity to converge and final registration quality. While CC gave the best results for the optimization of 𝑇 1 , the task for 𝑇 2 revealed to be more challenging. Therefore, in addition to these two metrics, we also evaluated the Mutual Information (MI) metric and the ANTs cross-correlation (ANTs CC). After some tests, we chose to use the gradient descent as an optimizer. Table 1 shows the results of this comparison on two animals. The final error is calculated on the resulting subtraction of the superimposed volumes, using only the voxels contained in the cranial skull mask. We define it as follows:

𝐸𝑟𝑟𝑜𝑟 = 𝑁 𝑒𝑟𝑟𝑜𝑟 𝑁 𝑡𝑜𝑡 (3)
Where 𝑁 𝑒𝑟𝑟𝑜𝑟 is the number of voxels composing the mask with an absolute intensity above a threshold 𝜀 = 3σ, σ being the intensity standard deviation among these voxels. 𝑁 𝑡𝑜𝑡 is the total number of voxels composing the mask.

As the table 1 shows, while the 4 methods produce comparable results in terms of quality, they differ when considering convergence speed. MSQ being less expensive to compute than both CC and MI, it is faster to converge. Given the fact there is no significant improvement using ANTs metric in this context, we opted for the MSQ metric defined as below:

𝑀𝑆𝑄(𝑓, 𝑚) = ∑ ( 𝑓 -𝑚 )² 𝑥𝑦𝑧 𝑛 (4)
Where 𝑓 and 𝑚 represent the vectors of the fixed and moving image intensities and n the total number of voxels.

K-edge subtraction and gold extraction

Once the volumes have been subtracted as defined in equation ( 2), we need to distinguish noise and artefacts from the AuNP-labelled cells. In order to do so, we begin with only keeping the parts corresponding to the brain by calculating the convex hull of the segmented cranial skull. We then subtract the dilated skull's mask leaving us with a limited number of structures, the biggest one corresponding to the brain. The next step consists in differentiating the gold NPs from the noise. We therefore apply a threshold defined as follows:

𝜀 𝐺𝑜𝑙𝑑 = 3𝜎 𝐵𝑟𝑎𝑖𝑛 (5)
𝜎 𝐵𝑟𝑎𝑖𝑛 being the standard deviation in the segmented brain. This operation is followed by a binary 3D opening with a 2 voxels radius sphere to remove the small structures that can be considered as noise. An example of result on rat 2 can be seen in Figure 5c.

Time process

Table 2 shows the computation time for the entire pipeline for the two rats presented above. The developed pipeline allows to have a total computation time in the same order as the acquisition time. The difference between the 2 rats is explained by the fact that rat 2 has moved less, only by a translation, and the algorithm tries nevertheless to improve the results by a rotation that is difficult to find and uses the maximum number of steps allowed in our implementation. Another explanation is the size of rat 2 cranial skull is bigger.

CONCLUSION

In this work we have presented a whole image analysis pipeline for the automatic segmentation of spectral phase contrast imaging. The developed method uses the specificities of the synchrotron phase contrast signal to improve the contrast in our images. After some tests using different metrics, the registration method uses a gradient descent as optimizer, trilinear interpolation and cross correlation and mean squared difference as metrics. Once the spectral images are registered, we compute the maps of gold concentration in order to track the gold nanoparticles-labelled cells. We presented results on two rats but we tested our method on a whole dataset of in vivo rats with a focal cerebral injury mimicking stroke. The algorithm proved to be robust and fast on big datasets (several gigabytes per rat). The main limitation of this study is the fact that we only tested our pipeline on a cerebral application but we will use and test this pipeline on another medical disease (osteoarthritis) in the near future.

Figure 1 .

 1 Figure 1. Combined Phase contrast and K-edge Imaging technique (A: Indices of refraction and absorption dependence in energy for water, bone and gold. B: Phase contrast imaging set-up).

Figure 3 .

 3 Figure 3. Intensity-based skull segmentation (A: Slice of the acquired volume, B: Histogram and its 3 intensity groups, C: Corresponding intensity regions (yellow: skull, red: jaws, green: gold)).

Figure 4 .

 4 Figure 4. An example of the importance of registration for K-edge subtraction (A: Slice of 𝑉 𝐴𝑏𝑜𝑣𝑒 , B: Slice of 𝑉 𝐵𝑒𝑙𝑜𝑤 , C: Slice of the resulting K-edge subtraction without registration, D: Slice of the resulting K-edge subtraction with registration). The negative concentrations are caused by non-superimposed elements (hair in C and D, bones, gold in C).

Figure 5 .

 5 Figure 5. Results of gold extraction (A: Slice of 𝑉 𝐴𝑏𝑜𝑣𝑒 , B: Slice of 𝑉 𝐵𝑒𝑙𝑜𝑤 , C: Slice of the segmented gold in the resulting K-edge subtraction, D: 3D rendering of the segmented skull (white) and segmented gold (yellow))

Table 1 .

 1 Comparison of 4 similarity metrics in terms of speed and quality. Mean Square Difference produces close to best results in the shortest period of time compared to other metrics.

			CC	MSQ	ANTs CC (r=2)	MI
	Rat 1	Time (s) Final Error	114 1.331%	71 1.316%	442 1.401%	143 1.328%
	Rat 2	Time (s) Final Error	365 0.7214%	258 0.7201%	867 0.7401%	428 0.7196%

Table 2 :

 2 Time process (seconds) of each step of the developed pipeline, measured on 2 rats.

		Skull Extraction	Registration	Subtraction	Gold Extraction	Total
	Rat 1	39.0s	208s	3.23s	80.1s	331s
	Rat 2	59.6s	419s	3.97s	92.9s	576s


Luke B Roberts

Corinna Schnoeller

Rita Berkachy

Matthew Darby

Jamie Pillaye

Menno J Oudhoff

Naveen Parmar

Claire Mackowiak

Delphine Sedda

Valerie Quesniaux

Bernhard Ryffel

Rachel Vaux

Kleoniki Gounaris

Sylvie Berrard

David R Withers

William G C Horsnell
email: wghorsnell@gmail.com

Murray E Selkirk
email: m.selkirk@imperial.ac.uk

Acetylcholine production by type 2 innate lymphoid cells promotes mucosal immunity to helminth parasites Authors

Innate lymphoid cells (ILCs) are critical mediators of immunological and physiological responses at mucosal barrier sites. While neurotransmitters can stimulate ILCs, the synthesis of small-molecule neurotransmitters by these cells has only recently begun to be appreciated. Type 2 innate lymphoid cells (ILC2s) are shown here to synthesise and release acetylcholine (ACh) during parasitic nematode infection. The cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin (IL)-33 and IL-25, and was augmented by IL-2 in vitro. Genetic disruption of ACh synthesis by murine ILC2s resulted in increased parasite burdens, impaired ILC2 proliferation, and reduced features of lung and gut barrier responses following Nippostrongylus brasiliensis infection. These data demonstrate a functional role for ILC2-derived ACh in expansion of ILC2s for maximal induction of type 2 immunity.

One-sentence summary

Synthesis of acetylcholine by type 2 innate lymphoid cells is important for optimal immune responses to helminth infection.

Introduction

Although acetylcholine (ACh) is best known as a small-molecule neurotransmitter, cholinergic signalling also regulates the immune system. This is best described in the cholinergic anti-inflammatory pathway (CAIP), in which sensory perception of inflammatory stimuli leads to a vagal reflex culminating in α7 nicotinic receptor (nAChR) subunit-dependent inhibition of TNF-α, IL-1β and IL-18 production by splenic macrophages [START_REF] Tracey | The inflammatory reflex[END_REF][START_REF] Wang | Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation[END_REF] . Definition of cells which synthesise ACh has been facilitated by the use of reporter mice to visualise expression of choline acetyltransferase (ChAT), the enzyme which synthesises ACh [START_REF] Tallini | BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons[END_REF]. CD4 + T cells expressing an effector/memory (CD44 + CD62L lo ) phenotype were identified as the source of ACh in the spleen responsible for signalling to macrophages in the CAIP (4) and B cell-derived ACh inhibited neutrophil recruitment during sterile endotoxaemia [START_REF] Reardon | Lymphocyte-derived ACh regulates local innate but not adaptive immunity[END_REF]. Additionally, CD4 + and CD8 + T cell expression of ChAT induced by IL-21 is essential for tissue trafficking required for T cell-mediated control of viral infection [START_REF] Cox | Choline acetyltransferase-expressing T cells are required to control chronic viral infection[END_REF]. Adaptive immunity is also regulated by ACh, and optimal type 2 effector responses to the nematode parasite Nippostrongylus brasiliensis require signalling through the M3 muscarinic receptor (mAChR) [START_REF] Darby | The M3 Muscarinic Receptor Is Required for Optimal Adaptive Immunity to Helminth and Bacterial Infection[END_REF].

Group 2 innate lymphoid cells (ILC2s) play an important role in initiating type 2 immune responses, producing cytokines such as IL-13 and IL-5 which drive allergic inflammation and immunity to helminth infection [START_REF] Neill | Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity[END_REF][START_REF] Campbell | ILC2s mediate systemic innate protection by priming mucus production at distal mucosal sites[END_REF]. ILC2s have recently been shown to be both positively and negatively regulated by neurotransmitters such as neuromedin U (NMU) [START_REF] Cardoso | Neuronal regulation of type 2 innate lymphoid cells via neuromedin U[END_REF][START_REF] Klose | The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation[END_REF][START_REF] Wallrapp | The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation[END_REF] and noradrenaline [START_REF] Moriyama | Β2-Adrenergic Receptor-Mediated Negative Regulation of Group 2 Innate Lymphoid Cell Responses[END_REF] , whereas group 3 innate lymphoid cells (ILC3s) upregulate lipid mediator synthesis in response to vagally-derived ACh [START_REF] Dalli | Vagal Regulation of Group 3 Innate Lymphoid Cells and the Immunoresolvent PCTR1 Controls Infection Resolution[END_REF].

Interestingly, ILCs expressing receptors responsive to neurotransmitters co-localise with neurons in mucosal tissues, forming what have been termed neuroimmune cell units (NICUs) [START_REF] Veiga-Fernandes | Neuronal-immune system cross-talk in homeostasis[END_REF]. Additionally ILC2s express the neuropeptide calcitonin gene-related protein, CGMP [START_REF] Nagashima | Neuropeptide CGRP Limits Group 2 Innate Lymphoid Cell Responses and Constrains Type 2 Inflammation[END_REF]. Recently, expression of tryptophan hydroxylase 1 (Tph1)the rate-limiting enzyme for the synthesis of the small-molecule neurotransmitter serotonin, as well as production of serotonin itself by ILC2s has been described [START_REF] Flamar | Interleukin-33 Induces the Enzyme Tryptophan Hydroxylase 1 to Promote Inflammatory Group 2 Innate Lymphoid Cell-Mediated Immunity[END_REF].

In this study we demonstrate that pulmonary ILC2s upregulate their capacity to synthesise and release ACh during infection with N. brasiliensis and show that the cholinergic phenotype of ILC2s is induced by the alarmin cytokines IL-33 and IL-25. Disruption of the ability of ILC2s to synthesise ACh by generation of Rora Cre+ Chat LoxP transgenic mice resulted in impaired immunity to N. brasiliensis, accompanied by reduced total expression of type 2 cytokines IL-5 and IL-13 and the mucins Muc5b and Muc5ac in the lung, in addition to altered intestinal barrier responses. These data demonstrate that production and release of ACh by ILC2s is an important factor in driving type 2 immunity.

Science Immunology

Manuscript Template Page 4 of 37

Results

ILC2s synthesise and release acetylcholine during type 2 immunity

The cholinergic phenotype of immune cells was monitored across the time course of a primary infection with N. brasiliensis using ChAT-eGFP BAC mice [START_REF] Tallini | BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons[END_REF]. From day 4 post infection (D4 p.i., immediately following the pulmonary migratory phase of parasite larvae) until at least D21 p.i., (long past the peak of the acute phase of infection-driven inflammation) the proportion and number of CD45 + cells in lung tissue which expressed ChAT (ChAT-eGFP + ) was increased compared to uninfected (naïve) controls (Figure 1A).

Analysis of ChAT-eGFP + leucocytes revealed that most of these cells were from lymphoid rather than myeloid lineages, as previously reported in other models and tissues (5) (Figure 1B). Of the populations screened, only ILC2s significantly upregulated the proportion of cells expressing ChAT-eGFP at an early time point (D4) in infection (Figure 1B).

As a cholinergic phenotype for ILC2s had not been previously described, we explored this further. Kinetic analysis of ChAT-eGFP expression by ILC2s in lung and bronchoalveolar lavage (BAL) samples during infection showed a significant increase by D4 p.i., peaking at D7, and remained elevated in both sites at D21.

The proportion of ILC2s which were ChAT-eGFP + was consistently much greater in BAL than in the lungs (Figure 1C, 1D). Real-time RT-qPCR confirmed that Chat expression in pulmonary ChAT-eGFP + ILC2s from infected ChAT-eGFP BAC mice was upregulated in comparison to ILC2 from uninfected ChAT-eGFP BAC animals, as well as to ChAT-eGFP neg ILC2, validating our reporter system (Figure 1E). HPLCmass spectrometry was used to verify that WT ILC2s synthesise and release ACh and showed that this was greatly enhanced during parasite infection (Figure 1F). In these experiments, cells were isolated from infected animals at D11 p.i. to maximise the number of ACh-producing ILC2s obtained. Additionally we observed that ChAT-eGFP + ILC2s had a significantly increased mean fluorescence intensity (MFI) for the IL-33 receptor subunit ST2 compared to ChAT-eGFP -cells at D4 and D7 p.i. (Figure 1G) and also for inducible T cell co-stimulator (ICOS) at D7 p.i. (Figure 1H), suggesting that ChAT expression is associated with ILC2 activation state.

A significant degree of heterogeneity exists amongst ILC2s, with subtypes such as tissue-resident 'natural' ILC2s (nILC2s) and tissue-infiltrating 'inflammatory' ILC2s (iILC2s) previously described and delineated on the basis of differential levels of phenotypic marker expression in the lung [START_REF] Huang | IL-25-responsive, lineage-negative KLRG1 hi cells are multipotential "inflammatory" type 2 innate lymphoid cells[END_REF]. Furthermore, the functions of these subtypes have physiological relevance with regards to anti-helminth immune responses such as pulmonary mucus production [START_REF] Campbell | ILC2s mediate systemic innate protection by priming mucus production at distal mucosal sites[END_REF]. To further characterise pulmonary ChAT-eGFP + ILC2s and probe whether these cells belonged to a defined subtype of ILC2s, we infected ChAT-eGFP BAC mice with N.

brasiliensis and analysed pulmonary ILC2s at D7 p.i, utilising an extended panel of phenotypic markers (Figure 2A). To assess whether ChAT-eGFP + ILC2s represented previously recognised nILC2 and iILC2 subsets, we used t-distributed stochastic neighbor embedding (t-SNE) analysis (omitting ChAT-eGFP expression as a component for clustering), to first identify populations of ILC2s (CD45 + Lineage-CD127 + ICOS + CD90 + cells expressing either or both ST2 and IL-17RB) which most represented conventional nILC2 (IL-17RB -ST2 + CD90 + Klrg1 lo/-) and iILC2 (IL-17RB + ST2 lo/-CD90 lo Klrg +/hi ) subsets (Figure 2A). We also identified additional clusters which we designated 'nILCa' and 'iILC2a' on the basis that these populations appeared to represent nILC2-like and iILC2-like cells in a higher state of cellular activation, given their differential expression of IL-17RB, but dual expression of ST2 and Klrg1, and higher expression of CD90. Given our previous observation that ChAT-eGFP expression in ILC2s appeared to correlate with cellular activation, we reasoned that these groupings may be relevant for comparative analysis.

Indeed, it appeared that the majority of ILC2s expressing high levels of ChAT-eGFP were located amongst these activated nILC2a and iILC2a populations (Figure 2B). Analysis was again carried out utilising t-SNE (with ChAT-eGFP expression incorporated into clustering), and ChAT-eGFP + clusters could be segregated into 3 distinct populations, designated C1-C3 (Figure 2C). Based on a combination of marker expression and comparative assessment of the ChAT-eGFP + clusters against the 4 pre-defined reference subtypes, population C1 appeared most similar to conventional nILC2 cells, whilst C2 shared the phenotypic profile of nILC2a, and C3 was most similar to iILC2a (Figure 2D). The greatest proportion of ChAT-eGFP + ILC2s

were represented by population C2, followed by C3, then C1 (Figure 2E). A similar analysis of marker expression of the very few ChAT-eGFP + cells in naïve ChAT-eGFP BAC lungs revealed that these cells clearly showed a nILC2-like profile, with no obvious differences in marker expression to that of total ChAT neg ILC2s, including ICOS and ST2 (Figure 2F, 2G, 2H). A different scenario was observed in infected mice however, with a disparate profile for total ChAT-eGFP + ILC2s relative to total ChAT neg ILC2s (Figure 2F, 2G, 2H), corroborating the findings of previous analyses (Figure 1G, 1H). ChAT-eGFP + ILC2s therefore do not appear to represent a singular ILC2 subtype during N. brasiliensis infection. One interpretation of these data may be that in infected, population C1 represents the ChAT-eGFP + nILC2-like cells found in naïve mice, while populations C2 and C3 represent nILC2-like and iILC2-like populations induced to express ChAT as a result of infection, supporting our hypothesis that ChAT-eGFP expression in ILC2s is predominantly associated with cellular activation.

To investigate whether ChAT-eGFP expression by ILC2s was solely a feature of pulmonary tissues, we examined ILC2s from mesenteric lymph nodes (MLNs) ChAT-eGFP + ILC2 were found in MLNs of both naïve and N. brasiliensis-infected mice (Figure 3A). However, as in pulmonary populations, the proportion of ILC2s expressing ChAT-eGFP increased as a result of infection (Figure 3B), as did the total numbers (Figure 3C). Interestingly, ChAT-eGFP + ILC2s in the MLNs of infected mice displayed a different phenotypic profile to ChAT-eGFP neg ILC2s, particularly evident when observing Klrg1 expression, which was restricted to ChAT-eGFP + cells, accompanied by higher expression of ST2, ICOS, and (generally) IL-17R (Figure 3D,3E,3F). This difference in marker expression was also apparent between ChAT-eGFP + and ChAT-eGFP neg ILC2s of naïve MLNs, although expression levels were much greater following infection, as expected (Figure 3E,3F). Given the difficulty in isolating viable leucocytes from the small intestinal lamina propria (siLP) of N. brasiliensis infected mice, we were not able to analyse ChAT-eGFP expression by flow cytometry from siLP ILC2 during infection. However, siLP ILC2s from naïve mice did not greatly express ChAT-eGFP (Figure 3G), indicating that siLP ILC2s do not constitutively display a cholinergic phenotype in the absence of infection, akin to our observations in naïve lungs. Overall, these data indicate that ChAT-eGFP expression by ILC2s is not limited to pulmonary populations, and further support association of the cholinergic phenotype with cellular activation.

Next, we tested if induction of the cholinergic phenotype in ILC2s was specific to parasite infection or a general feature of type 2 immunity. Mice exposed to extracts of Alternaria alternata, a fungal plant pathogen linked to exacerbation of asthma, develop rapid onset type 2-driven eosinophilic airway inflammation [START_REF] Mcsorley | Blockade of IL-33 release and suppression of type 2 innate lymphoid cell responses by helminth secreted products in airway allergy[END_REF].

ChAT-eGFP BAC and WT mice were dosed intranasally with Alternaria extract or phosphate buffered saline (PBS), culled 24 hours later and lung cells analysed for ChAT-eGFP expression. Successful induction of a type 2 response was confirmed by pulmonary eosinophilia (Figure S1A). Challenge with Alternaria induced small but significant increases in ChAT-eGFP expression in some lymphocyte populations, including CD4 + T cells and NKT cells, although expression in granulocytes was again unaffected (Figure S1B). However, as in nematode infection, the greatest proportional increase in ChAT-eGFP expression was observed in ILC2s (Figures S1C,S1D).

These data show that ILC2s have an inducible cholinergic phenotype, and that this is activated in response to parasitic nematode infection and exposure to a fungal allergen. This identifies ILC2s as the principle innate source of ACh during type 2 immune responses.

IL-25 and IL-33 induce the cholinergic phenotype of pulmonary ILC2s

As our data suggested that ChAT expression was associated with cellular activation, we investigated whether known activators of ILC2s could induce this phenotype. Ex vivo stimulation of CD45 + cells isolated from naïve ChAT BAC -eGFP reporter mice with IL-33, but not IL-7 enhanced ILC2 ChAT-eGFP expression, suggesting that activation through alarmin signalling pathways specifically drives the ILC2 cholinergic phenotype (Figure 4A). To explore this further, we dosed reporter mice intranasally with IL-33, IL-25 and thymic stromal lymphopoietin (TSLP), and analysed ChAT-eGFP expression on pulmonary ILC2s 24 hrs later. IL-25 and IL-33 both induced ChAT-eGFP expression on ILC2s, with IL-25 having the greatest effect; no effect was observed with TSLP (Figure 4B-C). Analysis of other leukocyte populations in the lung showed that activation of ChAT-eGFP expression by alarmins was only observed in ILC2s at the time point investigated (Figure 4D).

The observation that IL-25 appeared to promote greater upregulation of ChAT expression than IL-33, 24 hours after administration of recombinant cytokines, was interesting. Lung ILC2s predominantly express the IL-33 receptor in naïve animals at immunological baseline, while iILC2s expressing the IL-25 receptor are thought to migrate to the lung from sites such as the gut following tissue damage such as that caused by helminth infection [START_REF] Huang | IL-25-responsive, lineage-negative KLRG1 hi cells are multipotential "inflammatory" type 2 innate lymphoid cells[END_REF]. It is possible that administration of recombinant IL-25 mobilised ILC2s from outside the lungs to migrate to the pulmonary tract and that these cells contributed to the increase in ChAT-eGFP + ILC2s, although this is unlikely as it would have to happen within 24 hours. However, it is noteworthy that although the proportion of IL-17RB-expressing ILC2s in the lungs increases significantly following N.

RoRα-driven disruption of ChAT expression impairs pulmonary type 2 immunity to N. brasiliensis

To determine whether synthesis of ACh by ILC2s played a role in immunity to helminth infection, we generated Rora Cre+ Chat LoxP mice in which a portion of the coding domain of the Chat gene is floxed [START_REF] Lecomte | Selective disruption of acetylcholine synthesis in subsets of motor neurons: A new model of late-onset motor neuron disease[END_REF] and excised by Cre-recombinase expressed under the control of Rora regulatory elements (23) (Figure S2A-B).

The use of Rora Cre+ mice to selectively carry out gene deletion in ILC2s has been previously demonstrated [START_REF] Omata | Group 2 Innate Lymphoid Cells Attenuate Inflammatory Arthritis and Protect from Bone Destruction in Mice[END_REF]. Successful Chat deletion in Rora Cre+ Chat LoxP ILC2s was confirmed by PCR analysis and sequencing (Figure S2C, S2D, S2E). Infection of Rora Cre+ Chat LoxP and Chat LoxP littermate controls with N. brasiliensis revealed that although numbers of larvae recovered from the lungs were not significantly different between genotypes at day 2 post infection (p.i.), significantly higher intestinal worm burdens were observed at day 6 p.i. in Rora Cre+ Chat LoxP mice, indicating delayed parasite clearance in the absence of ILC2 ChAT expression (Figure 5A). Interestingly, Rora Cre+ Chat LoxP mice had reduced pulmonary eosinophilia compared to controls following N. brasiliensis infection, indicative of a suppressed type 2 immune response (Figure 5B).

Significantly reduced expression of Il5 and Il13 in total lung tissue of infected Rora Cre+ Chat LoxP samples, relative to controls supported this observation (Figure 5C). A primary role for IL-13 during the anti-helminth immune response is to drive goblet cell hyperplasia and mucin production at epithelial barrier sites including the lung, where the predominant gel-forming mucins secreted by goblet cells are Muc5b and Muc5ac [START_REF] Fahy | Airway Mucus Function and Dysfunction[END_REF].

Accordingly, infected Rora Cre+ Chat LoxP at D6 p.i. demonstrated significantly reduced expression of Muc5b and Muc5ac in total lung tissue (Figure 5D). PAS staining also revealed significantly reduced airway mucins in Rora Cre+ Chat LoxP lungs compared to the robust response observed in Chat LoxP airways (Figure 5E-F).

Impaired immunity to N. brasiliensis in Rora Cre+ Chat loxP mice is associated with defective intestinal barrier responses

To assess whether a defective response to N. brasiliensis infection following RoRα-mediated ChAT disruption was confined to pulmonary ILC2s and associated responses in the lung, we also assessed responses in the small intestine. Intestinal epithelial effector responses characteristic of type 2 immunity include goblet and tuft cell hyperplasia. In the small intestine, we observed a decrease in periodic acid-Schiff (PAS)-positive goblet cells comparing Rora Cre+ Chat LoxP samples to Chat LoxP controls (Figure 6A,6B).

Furthermore, we quantified tuft cells as analysed through immunofluorescent staining of doublecortin-like kinase 1 (Dclk1) (Figure 6C). We found that, although Rora Cre+ Chat LoxP mice did not have a statistically reduced number of tuft cells overall (Figure 6D), the ratio of cells present in villus to crypt regions was significantly lower in Rora Cre+ Chat LoxP mice (Figure 6E). In line with delayed worm expulsion (Figure 5A), these data reflect an inhibited onset of type 2 immunity in these animals.

ILC2-derived ACh promotes autocrine population expansion of ILC2s to facilitate optimal anti-

helminth type 2 immunity.

ILC2s are the major innate source of IL-13 during helminth infection and ILC2-derived IL-13 is critical for expulsion of N. brasiliensis and induction of mucin expression in response to helminth infections [START_REF] Neill | Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity[END_REF][START_REF] Campbell | ILC2s mediate systemic innate protection by priming mucus production at distal mucosal sites[END_REF][START_REF] Price | Systemically dispersed innate IL-13-expressing cells in type 2 immunity[END_REF].

Given our finding that RoRα-mediated disruption of Chat expression negatively impacted the type 2 immune response to N. brasiliensis infection, we next analyzed whether ILC2s themselves were affected by removing their capacity to synthesise ACh. Although total number of ILC2s in the lungs increased following N.

brasiliensis infection regardless of genotype, significantly fewer ILC2s were found in Rora Cre+ Chat LoxP lungs (Figure 7A). Of note, slightly fewer ILC2s were also observed in Rora Cre+ Chat LoxP lungs compared to

Chat LoxP lungs at baseline (Figure 7A) and the fold change for infection-induced increase in ILC2 numbers at this timepoint was not significantly different between genotypes (Figure S3A ILC2s. We also analysed the number of CD4 + lung T cells (Figure S4A), their proliferative capacity (Figure S4B, S4C), and expression of IL-13 (Figure S4D), but found that these parameters were unaffected by genotype, indicative of an ILC2-specific effect of Chat deletion in Rora Cre+ Chat LoxP mice.

Lymphocytes are known to express acetylcholine receptors (AChRs), although the full complement of muscarinic (mAChR) and nicotinic (nAChR) receptors expressed by ILC2s has not been defined to our knowledge [START_REF] Darby | The M3 Muscarinic Receptor Is Required for Optimal Adaptive Immunity to Helminth and Bacterial Infection[END_REF][START_REF] Galle-Treger | Nicotinic acetylcholine receptor agonist attenuates ILC2-dependent airway hyperreactivity[END_REF]. Using cDNA prepared from FACS-purified ChAT-eGFP + and ChAT-eGFP neg lung ILC2s from N. brasiliensis-infected ChAT-eGFP BAC mice, we observed expression of transcripts for multiple mAChRs in addition to the α7nAChR [START_REF] Galle-Treger | Nicotinic acetylcholine receptor agonist attenuates ILC2-dependent airway hyperreactivity[END_REF]. Interestingly, there appeared to be a degree of differential expression between ChAT-eGFP + and ChAT-eGFP neg with regards to AChR subtypes (Figure 7H demonstrating that MyD88-dependent signalling is not essential for this in ILC2s. A factor common to both signalling pathways, such as the signal transducer TRAF6, may be required for inducible ChAT expression in ILC2s. TSLP, which does not signal through either MyD88 or TRAF6-dependent pathways, did not induce ChAT expression in ILC2s when administered in vivo at the same dose. However, although TSLP has been reported to influence cutaneous ILC2 activation [START_REF] Kim | TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation[END_REF], most studies identify IL-25 and IL-33 as the major inducers of ILC2 responses in the lung and gut [START_REF] Price | Systemically dispersed innate IL-13-expressing cells in type 2 immunity[END_REF][START_REF] Kim | Innate lymphoid cells responding to IL-33 mediate airwayhyperreactivity independent of adaptive immunity[END_REF].

IL-2 was also shown to induce ChAT expression, both by itself or in combination with IL-25 and IL-33. IL-2 is a critical regulator of ILC2s, driving cell survival and proliferation, and augmenting type 2 cytokine production [START_REF] Roediger | IL-2 is a critical regulator of group 2 innate lymphoid cell function during pulmonary inflammation[END_REF]. Thus, the cholinergic phenotype of ILC2s is clearly induced by a number of stimuli which activate these cells. Recently, neuropeptides such as NMU, vasoactive intestinal peptide (VIP) and CGRP and the small molecule neurotransmitter serotonin have also been shown to regulate ILC2 activation and effector activity (10-12, 16, 17, 31). If these molecules can also induce or modulate ChAT expression in ILC2s, this raises the possibility of bi-directional neuroimmune communication involving ILC2s and AChresponsive neurons within NICUs.

Impairment of ACh synthesis by ILC2s resulted in a lower number of cells, a deficit in ILC2-derived effector cytokine production and significant restriction of anti-helminth type 2 responses in the lungs and small intestine, tissue sites physiologically relevant to the parasite's life cycle. One interpretation of this may be that ILC2-derived ACh acts as an autocrine signal to aid population expansion. One way this could hypothetically occur is if ACh acts to promote expression of autocrine survival factors such as IL-9 (32), the expression of which has been shown to be promoted by neuropeptide signaling in ILC2s [START_REF] Klose | The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation[END_REF]. The labile nature of ACh, due to high levels of circulating butyrylcholinesterase in tissue fluids [START_REF] Lockridge | Review of human butyrylcholinesterase structure, function, genetic variants, history of use in the clinic, and potential therapeutic uses[END_REF] makes it likely that ILC2-derived ACh will function over relatively short distances, as would be the case during autocrine signaling. In support of this, we demonstrated that ILC2s express a range of nicotinic and muscarinic ACh receptors, and expression of the α7 nAChR by ILC2s has also been previously described [START_REF] Galle-Treger | Nicotinic acetylcholine receptor agonist attenuates ILC2-dependent airway hyperreactivity[END_REF]. In vitro culture of ILC2s with 4-DAMP led to a reduction in ILC2 proliferative capacity. Thus it is plausible that autocrine cholinergic signalling in ILC2s operates through mAChRs in order to promote their own expansion.

Although 4-DAMP is frequently quoted as M3/M1-selective, studies on rat and human receptors show that it has potent effects on M1, M3, M4 and M5 receptors (Ki less than 1 nM), and also good activity against M2 (Ki 4-7 nM). [START_REF] Moriya | Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland[END_REF][START_REF] Dorje | Antagonist binding profiles of five cloned human muscarinic receptor subtypes[END_REF]. Thus, at the concentration used in our experiments, 4-DAMP would be expected to antagonise all mAChR subtypes.

Anticholinergics in the form of mAChR antagonists such as tiotropium are widely used to treat asthma and chronic obstructive pulmonary disease [START_REF] Gosens | Muscarinic receptor signaling in the pathophysiology of asthma and COPD[END_REF]. These antagonists alleviate bronchoconstriction and mucus production, and are well documented to ameliorate allergen-induced airway inflammation and remodelling [START_REF] Bos | Inhibition of allergen-induced airway remodelling by tiotropium and budesonide: A comparison[END_REF][START_REF] Bosnjak | Tiotropium bromide inhibits relapsing allergic asthma in BALB/c mice[END_REF], which may be accompanied by reduced type 2 cytokine production [START_REF] Ohta | Effect of tiotropium bromide on airway inflammation and remodelling in a mouse model of asthma[END_REF]. In contrast, a recent report indicated that an α7-selective nAChR agonist reduced ILC2 effector function and airway hyperreactivity in an Alternaria allergic inflammation model [START_REF] Galle-Treger | Nicotinic acetylcholine receptor agonist attenuates ILC2-dependent airway hyperreactivity[END_REF]. It is thus likely that signalling through different AChRs on

ILC2s can result in disparate outcomes, and this could be affected by altered receptor expression under differing physiological conditions.

A surprising observation was that ChAT expression by ILC2s was maintained long after helminth eradication from the host (D21 p.i.). In addition to critical type 2 effector functions, ILC2s play important direct and indirect roles in promotion of wound healing and tissue repair [START_REF] Monticelli | Innate lymphoid cells promote lung tissue homeostasis following acute influenza virus infection[END_REF][START_REF] Molofsky | Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages[END_REF]. It will be interesting to determine whether ACh production by ILC2s plays a role during later, pro-repair activities in addition to the acute inflammatory phase of infection.

A limitation of our study is that, despite the critical role of RoRα in ILC2 development [START_REF] Wong | Transcription factor RORα is critical for nuocyte development[END_REF][START_REF] Halim | Retinoic-Acid-Receptor-Related Orphan Nuclear Receptor Alpha Is Required for Natural Helper Cell Development and Allergic Inflammation[END_REF], expression of RoRα is not confined to ILC2s [START_REF] Cook | Retinoic Acid-Related Orphan Receptors (RORs): Regulatory Functions in Immunity, Development, Circadian Rhythm, and Metabolism[END_REF]. Therefore, despite the fact that the Rora Cre+ mouse has previously been used to successfully facilitate gene deletion in ILC2s in vivo [START_REF] Omata | Group 2 Innate Lymphoid Cells Attenuate Inflammatory Arthritis and Protect from Bone Destruction in Mice[END_REF], it is feasible that deletion in other cell types may also have occurred, and that this may have contributed to our observations. However, proliferation and Th2 cytokine production by CD4 + T cells, which are also known to express this transcription factor, were not affected during N. brasiliensis infection of Rora Cre+ Chat LoxP mice.

Helminths have evolved sophisticated strategies to promote survival in their hosts, targeted at key drivers of anti-parasite immunity [START_REF] Maizels | Modulation of Host Immunity by Helminths: The Expanding Repertoire of Parasite Effector Molecules[END_REF]. Secretion of acetylcholinesterases (AChEs) by parasitic nematodes has been postulated to promote parasite persistence via inhibition of cholinergic signalling in relation to the 'weep and sweep' response, characterised by intestinal smooth muscle contraction and fluid secretion from epithelial cells [START_REF] Selkirk | Functional genomics of nematode acetylcholinesterases[END_REF]. Our current study demonstrates that production of ACh by ILC2s is a key factor in population expansion, driving maximal type 2 immunity and mucin expression, suggesting that hydrolysis of ACh by secreted AChEs may also act to suppress this to a level which allows parasite establishment within the host. Cytokines. Recombinant murine cytokines were purchased from R&D (IL-25, TSLP) or Peprotech (IL-33, IL-2, IL-7) and used at 50 ng ml -1 in vitro or administered in 50 µl doses at 10 µg ml -1 in vivo as indicated.

Materials and Methods

Animals

Tissue preparation. For isolation of bronchoalveolar cells, lungs were lavaged twice in a total of 2 ml PBS with 0.2% BSA and 2 mM EDTA. Erythrocytes were lysed, leukocytes resuspended and counted. For lung single cell suspensions, lungs were perfused via cardiac puncture with 10 ml PBS then infused with 1.5 ml PBS containing 5 mg ml -1 dispase II neutral protease (Sigma) via the trachea. The thymus and lung-draining lymph nodes were removed, lungs ligatured, removed into 1.5 ml digest solution, incubated at room temperature for 25 min, then for a further 30 min at 37⁰C. Lungs were mechanically dissociated in Dulbecco's Minimal Essential Medium (DMEM) with 25 mM HEPES and 100 U ml -1 DNAse I (Sigma), and incubated at room temperature for 10 min. Samples were passed through 100 μm cell strainers and erythrocytes lysed.

Single cell preparations of mesenteric lymph nodes were generated by mechanical dissociation through a 40 µm cell strainer followed by standard erythrocyte lysis. Small intestinal lamina propria (siLP) leucocytes were isolated with EDTA-based stripping of the intestinal epithelial layer followed by tissue digestion using collagenase-D (0.5 mg ml -1 ), Dispase-II (1.5 mg ml -1 ) and 10 µg ml -1 DNAse I in HBSS without Mg 2+ or Ca 2+ + 2% FCS. A 40-80% Percoll gradient separation was used to isolate a leucocyte enriched siLP sample.

Flow cytometry and cell sorting. Single cell suspensions were stained with fixable viability dyes (Invitrogen), then treated with rat anti-mouse CD32/CD16 (FcBlock, BD Biosciences), washed, then stained for extracellular markers using fluorophore conjugated monoclonal antibodies (eBioscience, Miltenyi Biotec or Biolegend). For intracellular staining, cells were fixed for 30 min at room temperature, then permeabilised using the FoxP3/transcription factor staining buffer kit (eBioscience) and stained with fluorochromeconjugated antibodies. Unstained samples and fluorescence minus one controls were used as appropriate.

When analyzing eGFP fluorescence from ChAT-eGFP BAC reporter mouse cells, WT (C57BL/6J) cells were used as negative controls to set eGFP gates. Samples were analysed on a BD LSR Fortessa™ analyser. For FACS sorting of ILC2s, lung tissue was processed to a single cell suspension as described, the lineage negative population enriched by magnetic activated cell sorting, depleting other cells via a PE-conjugated lineage cocktail (Miltenyi Biotec), then ILC2s sorted on a BD FACS ARIA III cell sorter.

Immunophenotyping of leucocyte populations. Unless otherwise stated, leucocyte populations were identified by flow cytometry by gating live cells, followed by single cell and CD45 + gating, and then using the following markers: ILC2; lineage -CD90 + ICOS + ST2 + CD127 + . T cells; CD19 -CD3ε + . CD4 + T cells; CD19 - CD3ε + DX5 -CD4 + CD8α -. CD8 + T cells; CD19 -CD3ε + DX5 -CD4 -CD8α + . B cells; CD19 + CD3ε -B220 + . γδ T cells; CD19 -CD3ε + DX5 -CD4 -CD8α -GL3 + . NK-T cells (NKT); CD19 -CD3ε + DX5 + . Natural Killer cells (NK); CD19 -CD3ε -DX5 + FcεR1 -. Neutrophils; CD11b + SIGLEC-F -GR-1 hi CD11c lo Eosinophils;

CD11b + SIGLEC-F + GR-1 lo CD11c lo . Basophils; CD19 -CD3ε -DX5 + FcεR1 + . The lineage panel consisted of antibodies to CD3, CD4, CD8, B220, CD19, TER119, CD49b, FcεRI and CD11b. Additional markers analysed for phenotyping and functional analysis were assessed using antibodies against Klrg1, IL-17RB, IL-5 and IL-13.

Ki67 and intracellular cytokine staining. To assess proliferative capacity of cells directly ex vivo, samples

were processed to single cell suspension as described and rested in cDMEM (DMEM + 10% FCS, + 2mM L-glutamine + 100U ml -1 Penicillin + 100ug ml -1 streptomycin) for 1 hour at 37 o C/5%CO2, before extracellular staining, fixing and permeabilization as detailed. Intracellular Fc receptor blocking followed by anti-Ki67 staining was then carried out with fluorophore conjugated mAbs in permeabilization buffer. To assess cytokine production, single cell suspensions were diluted to 5x 10 6 cells ml -1 in cDMEM and either stimulated for 4 hrs at 37 o C/5% CO2 with 1 ug ml -1 PMA/100 ng ml -1 ionomycin with 1x Brefeldin-A (GolgiPlug, BD Biosciences) + 1μM Monensin ( (Sigma) or left unstimulated (golgi -inhibitors alone).

Samples were stained, fixed and permeabilized as described and intracellular staining for Fc receptor blocking followed by fluorophore conjugated mAbs against IL-5 and IL-13 was carried out in permeabilization buffer.

ILC2 in vitro culture and proliferation analysis with acetylcholine receptor antagonists. Female C57BL/6J mice aged 6-8 weeks were infected with N. brasiliensis and lungs were processed and ILC2 FACS sorted at between D5 to D9 p.i. as described. For each experimental run, lungs from 5 mice were pooled and sorted as a single sample and isolated ILC2 were then split equally between experimental treatment conditions. Cells were cultured for 72 hours at 37 o C / 5% CO2 in U bottom 96 well plates (Greiner, Cellstar) in 200 μl of cRPMI (RPMI + 10% FCS + 2 mM L-glutamine + 100 U ml -1 Penicillin + 100 ug ml -1 streptomycin) containing 50 ng ml -1 recombinant human IL-2 (Biolegend) and 50 ng ml -1 recombinant murine IL-7 (Biolegend) and either 10 μM 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) (Abcam), 10 μM mecamylamine hydrochloride (mecamylamine) (Sigma), or an equal volume of drug vehicle (dH20 and DMSO) (vehicle control). Finally, cells were stained as described, including re-staining using the extracellular ILC2 marker panel used to initially sort the cells (to ensure purity of the cultured population), followed by intranuclear Ki67 staining. Samples were analysed on a BD LSR Fortessa™ analyser.

Endpoint PCR. Total RNA was extracted by TRIzol/chloroform phase-separation and DNAse-1 treatment.

Prior to RNA extraction, mouse brain tissue (used as a for positive control for all acetylcholine receptor subunit expressions) was homogenised using a Tissuelyser II (Qiagen). Reverse transcription (RT) of RNA was carried out in a 20 μl reaction volume with 50 ng RNA using the Superscript III reverse transcriptase Histology and PAS staining. Lung or small intestinal tissues were harvested, fixed in 10% buffered formalin, paraffin embedded and sectioned using standard techniques. Sections were stained with periodic acid-Schiff's and Haematoxylin and Eosin reagents, photographed at 40x or 100x magnification using a Zeiss Primo Star microscope and analysed using Image J to determine the Histological Mucus Index (HMI) by established methods [START_REF] Horsnell | IL-4Rα-Associated Antigen Processing by B Cells Promotes Immunity in Nippostrongylus brasiliensis Infection[END_REF]. Lung sections were analysed analysed using Image J. Briefly, lung images were overlaid with a standard grid (2000 Units) and the number of grid units containing PAS positive epithelial cells were divided by all units containing epithelial cells to establish the Histological Mucus index (HMI). Intestinal images were analysed with ImageJ and overlaid with a standard grid (1500 Units); 15 crypt-villus segments were selected and the percentage of PAS-positive units per crypt-villus segment were divided by total units in each crypt-villus to establish HMI.

Immunofluorescence. Jejunal sections were paraffin embedded and sectioned according to standard techniques. For immunofluorescent staining, tissue slides were incubated at 60°C for 30 min. Next, paraffin was removed in Neo-clear (Sigma-Aldrich) by washing twice for 5 min. Then, tissue was rehydrated in decreasing ethanol concentrations (2x 100%, 1x 95%, 1x 80 %, 1x 70 %, (all 3 min) and then in distilled water. Subsequently, tissue slides were heated and boiled in pH 6 citrate buffer for 15 min using a microwave. The citrate buffer was cooled to room temperature for 20 min, and slides were washed with distilled water. Next, tissues sections were marked using a hydrophobic pen (PAP pen, ab2601, Abcam) to prevent leakage. Tissue sections were incubated with blocking buffer (1% BSA, 2% normal goat serum, 0.2% Triton X-100 in PBS) for 1 hour at room temperature in a humified chamber. Next, slides were incubated with primary antibody for anti-DCLK1 (Abcam, ab31704) in antibody dilution buffer at 1:250 (0.5% bovine serum albumin, 1% normal goat serum, 0.05% Tween 20 in PBS) at 4°C overnight in a humified chamber. The next day, slides were washed 3 times in 0.2% Triton X-100 prepared in PBS for 10 min each. Slides were then incubated with the secondary antibody (1:500, Goat anti-Rabbit IgG Alexa Fluor 488, Invitrogen, A-11034,) and DAPI (1:1000) for 1 hour at room temperature in a dark humified chamber.

After incubation, slides were washed 3 times with 0.2% Triton X-100 prepared in PBS for 10 min each.

Finally, slides were washed with distilled water and were mounted using Fluoromount G medium (ThermoFisher Scientific) using cover slips. All the images were acquired with 20x and 40x objectives using a ZEISS confocal microscope LSM 880 and DCLK1 (green) cells were quantified for ≥30 crypt-villus pairs per mouse.

Statistical analysis. Flow cytometry data was analysed and t-SNE analysis was conducted using FlowJo software (Treestar). Graphs and statistical tests were carried out using Graphpad prism software (Graphpad).

  brasiliensis infection (Figure4E, 4F), around a fifth of ILC2s from the lungs of naïve mice (those found in naïve ChAT-eGFP BAC mice used in our study) expressed IL-17RB as well as ST2 (Figure4E, 4F), and thus have the capacity to respond to administration of exogenous IL-25.To assess the capacity of lung-resident ILC2s to upregulate ChAT-eGFP in response to exogenous alarmin cytokine treatment, we isolated CD45 + cells from the lungs of naïve ChAT BAC -eGFP reporter mice by FACS and stimulated them in vitro with combinations of recombinant cytokines and assayed ChAT-eGFP expression by ILC2s 24 hrs later. IL-2 is also known to promote proliferation and cytokine production by ILC2s[START_REF] Mirchandani | Type 2 innate lymphoid cells drive CD4+ Th2 cell responses[END_REF][START_REF] Oliphant | MHCII-mediated dialog between group 2 innate lymphoid cells and CD4+ T cells potentiates type 2 immunity and promotes parasitic helminth expulsion[END_REF] and so we also examined the capacity for IL-2 to upregulate ChAT-eGFP by the cells.Stimulation with IL-25 and IL-33 both enhanced ChAT-eGFP expression. However, IL-2 also induced ChAT-eGFP expression by ILC2s and moreover, an additive effect of stimulation with IL-2 and either IL-33 or IL-25 was observed (Figure4G-H).

  ), possibly indicative of a homeostatic requirement for Chat by ILC2s. A similar finding was made in the MLNs of infected Rora Cre+ Chat LoxP mice, where significantly fewer ILC2s were observed in comparison to infected controls (Figure S3B). Although ILC2s from Rora Cre+ Chat LoxP mice could still express IL-5 and IL-13 (Figure S3C-S3G) and upregulated cytokine expression in response to infection on a similar per-cell basis to Chat LoxP ILC2s (Figure S3E, S3G), the overall number of IL-13 and IL-5 expressing ILC2s was significantly reduced in infected Rora Cre+ Chat LoxP lungs at day 6 p.i. (Figure 7B). Ki67 staining of ILC2s revealed that Rora Cre+ Chat LoxP ILC2s were less proliferative than Chat LoxP ILC2s following infection (Figure 7C, 7D), resulting in a significantly smaller pool of proliferative ILC2s overall (Figure 7E). Additionally, expression of ICOS was significantly reduced on Rora Cre+ Chat LoxP ILC2s following infection, indicative of a decreased ILC2 activation state (Figure 7F, 7G). Similar observations were made for activation markers ST2 and ICOS on ILC2s in the MLNs of infected Rora Cre+ Chat LoxP (Figure S3H, S3I), indicating that the effects caused by prevention of ACh synthesis were not confined to pulmonary

  , 7I). In order to determine whether ACh might act as an autocrine factor to influence proliferation and activation of the cells, we isolated WT ILC2s from the lungs of N. brasiliensis-infected C57BL/6J mice and cultured them in vitro with IL-7 and IL-2 alone or in the presence of the mAChR antagonist 1,1-dimethyl-4diphenylacetoxypiperidinium iodide (4-DAMP) or the nAChR antagonist mecamylamine. Addition of 4-DAMP significantly restricted the proliferative capacity of the cells, while mecamylamine had no effect when compared with vehicle-treated control cultures (Figure7J, 7K, 7L). These data suggest that activation-induced ACh synthesis by ILC2s plays a role in mAChR-mediated autocrine promotion of ILC2 proliferation and population expansion.In summary these data support the hypothesis that ILC2-derived ACh plays a critical role in mounting robust type 2 immunity and control of N. brasiliensis infection.DiscussionILC2s play a pivotal role in translating epithelial cell cytokine production into robust type 2 immune responses. Here we show that in addition to the cytokines IL-13 and IL-5, production of ACh by ILC2s is a requirement for optimal type 2-driven immunity to N. brasiliensis. The alarmin cytokines IL-25 and IL-33 were demonstrated to upregulate ChAT-eGFP expression by ILC2s both in vivo and in vitro. As expression of ChAT by B cells is induced by MyD88-dependent Toll-like receptor signalling (5), we hypothesised that IL-33 might regulate the ILC2 cholinergic phenotype, as members of the IL-1 family such as IL-33 also signal through this adapter protein. However, IL-25 was also a major regulator of ChAT expression,

  protocol (Invitrogen) according to the manufacturer's instructions. Polymerase chain reaction (PCR) was carried out in a 20 μl reaction volume containing: 0.25 pmol forward and reverse primers, 1.25 mM dNTPs, 0.5 U Taq polymerase (New England Biolabs), 1x Thermopol reaction buffer (New England Biolabs), 2 µl cDNA and dH20. PCR products were visualised by agarose gel electrophoresis on a gel of appropriate agarose percentage (1-3%) made with a standard Tris-acetate-EDTA buffer (pH 8.0), utilising GelRed ® nucleic acid gel stain (Biotium) and a GelDOC-IT TS imaging system (UVP). Primers used for endpoint PCR are as follows: Chrm1: 5'-GGACAACAACACCAGAGGAGA-3'/5'-CGAGGTCACTTTAGGGTAGGG-3' Chrm2: 5'-TGAAAACACGGTTTCCACTTC-3'/5'-GATGGAGGAGGCTTCTTTTTG-3' Chrm3: 5'-TTTACATGCCTGTCACCATCA-3'/5'-ACAGCCACCATACTTCCTCCT-3' Chrm4: 5'-TGCCTCTGTCATGAACCTTCT-3'/5'-TGGTTATCAGGCACTGTCCTC-3' Chrm5: 5'-CTCTGCTGGCAGTACTTGGTC-3'/5'-GTGAGCCGGTTTTCTCTTCTT-3' Chrna1: 5'-GACCATGAAGTCAGACCAGGA-3'/5'-TTAGCTCAGCCTCTGCTCATC-3' Chrna2: 5'-TGAGGTCTGAGGATGCTGACT-3'/5'-AGAGATGGCTCCAGTCACAGA-3' Chrna3: 5'-GTTGTCCCTGTCTGCTCTGTC-3'/5'-CCATCAAGGGTTGCAGAAATA-3' Chrna4: 5'-AGATGATGACGACCAACGTGT-3'/5'-ATAGAACAGGTGGGCTTTGGT-3' Chrna5: 5'-TGGGCCTTGCAATATCTCAGT-3'/5'-TGACAGTGCCATTGTACCTGA-3' Chrna7: 5'-TCAGCAGCTATATCCCCAATG-3'/5'-CAGCAAGAATACCAGCAAAGC-3' Chrnb1: 5'-CTCACTGTGTTCTTGCTGCTG-3'/5'-GAGTTGGTCTCTCTCGGGTTT-3' electrophoresis, and bands corresponding to the WT allele (2076 bp) and the deleted allele (546 bp) were excised, purified by standard procedures, and sequenced by Eurofins Genomics. Detection of acetylcholine release. FACS-purified ILC2s were incubated at 37⁰C for 30 min in 96 well round-bottomed plates (10 5 cells in 150 μl), centrifuged, supernatants removed, the AChE inhibitor BW284C51 (Sigma) added at 10 µM and samples stored at -80 o C until analysis by HPLC-mass spectrometry. Control samples were spiked with 50 nM internal standard (acetylcholine -1,1,2,2,-D4 chloride, QMX laboratories).

Figure S1 .Figure S2 .

 S1S2 Figure S1. Pulmonary ILC2s acquire a cholinergic phenotype following exposure to Alternaria

Figure S3 .

 S3 Figure S3. Pulmonary ILC2 cytokine measurements and assessment of numbers and activation markers of mesenteric lymph node ILC2s in Chat LoxP and Rora Cre+ Chat LoxP mice.

Figure S4 .

 S4 Figure S4. Pulmonary CD4 + T cell numbers and capacity for Th2 cytokine expression remains intact in Rora Cre+ Chat loxp mice

Figure 1 .

 1 Figure 1. Pulmonary ILC2s acquire a cholinergic phenotype associated with an enhanced activation state during infection with Nippostrongylus brasiliensis. A) Proportion and total number of CD45 + leucocytes expressing ChAT-eGFP in the lungs of naïve ChAT BAC -eGFP mice or animals infected with N.

Figure 2 .

 2 Figure 2. ChAT-eGFP + ILC2s display a range of phenotypes which support positive association of

mice. D )

 D Representative histogram overlays for the indicated marker expressions of total ChAT-eGFP + and ChAT-eGFP neg MLN ILC2s of naïve and infected ChAT-eGFP BAC mice at D7 p.i.. E) Mean fluorescence intensity (MFI) of ST2 expressed by total ILC2 and ChAT-eGFP negative and ChAT-eGFP + ILC2s in the MLNs of naïve and infected mice as in (D). F) As for (E) but for ICOS geometric MFI (gMFI). G) Representative flow cytometry plot of ChAT-eGFP expression by small intestinal lamina propria (siLP) ILC2s in naïve ChAT BAC -eGFP animals. Gate number represents proportion of ILC2 parental gate. Data shown is representative of observations and population comparisons made in n = 3 male ChAT-eGFP BAC mice in both naïve and infected treatment groups (N = 1), apart from data shown in (G) which is representative of results in n = 2 mice. Data shown as mean + SEM and analysed by Shapiro-Wilk normality testing followed by Welch's t-test , *p<0.05, *p<0.01, ***p<0.001, ****p<0.0001.

Figure 4 .

 4 Figure 4. The cholinergic phenotype of pulmonary ILC2s is induced by IL-25 and IL-33 and is

Figure 5 .

 5 Figure 5. RoRα-driven disruption of ChAT expression impairs pulmonary type 2 immunity and

Figure 6 .

 6 Figure 6. Impaired immunity to Nippostrongylus brasiliensis in Rora Cre+ Chat loxP mice is associated with

  and parasite infection. This study was approved by the Animal Welfare Ethical Review Board at Imperial College London and was licensed by and performed under the UK Home Office Animals (Scientific

	Procedures) Act Personal Project Licence number 70/8193: 'Immunomodulation by helminth parasites'.
	C57BL/6J mice, aged 6-8 weeks old were purchased from Charles River. ChAT-eGFP BAC (3) were purchased

from Jackson Laboratories and subsequently bred in-house. Chat LoxP mice were generated as previously described

[START_REF] Lecomte | Selective disruption of acetylcholine synthesis in subsets of motor neurons: A new model of late-onset motor neuron disease[END_REF]

and were backcrossed to F6-F10 generations on a B6 background with Rora Cre+ (23) a kind gift from Andreas Zembrzycki (Salk Institute, La Jolla, CA) to generate the Rora Cre+ Chat Loxp mice used in this study. Mice were infected with N. brasiliensis by sub-cutaneous (s.c.) inoculation with 500 infective larvae and parasites maintained by established methods

[START_REF] Camberis | Current Protocols in Immunology[END_REF]

.

Murine model of allergic airway inflammation. Extracts of Alternaria alternata were obtained as a gift from Henry McSorley (University of Edinburgh) or purchased as lyophilised protein extract from Greer

Laboratories (USA). Mice were lightly dosed with isoflurane before intranasal administration with 50 μg A. alternata extract in a final volume of 50 µl PBS. Mice were exposed to a single dose of A. alternata for 24 hours. Control animals were dosed with 50 μl PBS in the same schedule.

Mesenteric lymph node ILC2s upregulate expression of ChAT following infection with Nippostrongylus brasiliensis. A)

  Contributions of populations C1, C2, C3 to total ChAT-eGFP + ILC2 present in the lungs of ChAT-eGFP BAC mice at D7 p.i, expressed as the contributing proportion of each population to the total. F) Representative histogram overlays for the indicated marker expressions of total ChAT-eGFP + and ChAT-eGFP neg ILC2s (identified as live, single cells, CD45 + Lineage neg CD127 + ICOS + CD90 + and expressing either or both ST2 and/or IL-17RB) present in the lungs of naïve and infected ChAT-eGFP BAC mice at D7 ChAT-eGFP BAC mice in both naïve and infected treatment groups. N=1. Data shown as mean + SEM and analysed by Shapiro-Wilk normality testing followed by Welch's t-test , *p<0.05, **p<0.01. n.s. = nonsignificant (p>0.05). Representative flow cytometry gating indicating identification of total ILC2s and ChAT-eGFP + and ChAT-eGFP neg ILC2s from the mesenteric lymph nodes (MLNs) of naïve and infected ChAT-eGFP BAC mice at D7 p.i. N. brasiliensis -infected C57BL/6J control sampled included to indicate ChAT-eGFP + gating control. B) Quantification of ChAT-eGFP+ ILC2s from naïve and D7 p.i. ChAT-eGFP BAC mice expressed as a proportion of total ILC2s as shown in (A). C) Quantification of the number of total, ChAT-eGFP + and ChAT-eGFP neg ILC2s from MLNs of naïve and D7 p.i. ChAT-eGFP BAC

	-CD90 hi ), inflammatory ILC2 (iILC2: IL17RB + ST2 lo Klrg1 + CD90 lo ) and nILC2/iILC2 with an activated-like phenotypic profile (nILC2a: IL17RB -ST2 + Klrg1 + CD90 + , iILC2a: IL17RB + ST2 + Klrg1 + CD90 Figure 3.

+ ) within the lungs of N. brasiliensis (Nb)-infected ChAT-eGFP BAC mice at day 7 post infection (D7 p.i.). Clustering analysis was conducted using t-distributed stochastic neighbour embedding (t-SNE), with the parameter of ChAT-eGFP expression omitted, carried out on total lung ILC2s (identified as live, single cells, CD45 + Lineage neg CD127 + ICOS + CD90 + and expressing either or both ST2 and/or IL-17RB) with indicated populations identified on the basis of various phenotypic markers and marker expression levels as indicated by the heatmap plots shown (right). B) Heatmap plot showing locations of greatest ChAT-eGFP expression amongst ILC2 clusters as defined through t-SNE in (A). C) Clustering analysis carried out by t-distributed stochastic neighbour embedding (t-SNE) as in (A) but with the parameter of ChAT-eGFP expression included (left) and heatmap plot of ChAT-eGFP expression to identify ChAT + cell clusters, designated C1, C2 and C3 (right). D) Representative histogram overlays of indicated marker expression for the indicated populations identified within a given biological samples. E) p.i.. G) Mean fluorescence intensity (MFI) of ST2 expressed by total ILC2 and ChAT-eGFP negative and ChAT-eGFP + ILC2s in the lungs of naïve and infected mice as in (F). H) As for (G) but for ICOS geometric MFI (gMFI). Data shown is representative of observations and population comparisons made in n = 3 male

Acknowledgments

General: We thank Mark Bennett for mass spectrometric determination of ACh, Henry McSorley for the kind gift of Alternaria alternata extract, and Jane Srivistava and Catherine Simpson for technical assistance with flow cytometry-assisted cell sorting. Funding: We thank the Wellcome Trust and the BBSRC for supporting this work through a PhD studentship to LBR (097011) and a project grant to MES and DW (BB/R015856/1). RV was supported by a Medical Research Council PhD studentship (MR/J500379/1), and RB was initially supported by a scholarship from the Association Philippe Jabre (apj.org.ib). Royal Society International Exchange grant: IES\R1\180108,

Data and materials availability: If data are in an archive, include the accession number or a placeholder for it. Also include any materials that must be obtained through an MTA.


L Tchang-Tchong

P Faure-Catteloin

C Lorgeoux

E C Gaucher

R Michels

EVALUATION OF ERODED MESOZOIC FORMATIONS IN NORTHERN ALSACE (FRANCE) USING ORGANIC GEOCHEMISTRY AND BASIN THERMAL MODELLING

Introduction

The Upper Rhine Graben (URG) is a Cenozoic intracontinental rift, which extends partly in France, Germany and Switzerland, 300 km long and 30-40 km wide [START_REF] Schumacher | Upper Rhine Graben: role of preexisting structures during rift evolution[END_REF]. The sedimentary basin is NNE/SW trending and its current geometry corresponds to a set of asymmetrical and antithetical halfgraben, connected by transfer zones using structures inherited from the Paleozoic. Pre-rift sedimentary filling is composed of a serie of Triassic to Middle Jurassic formations separated from syn-rift Cenozoic deposits by a major unconformity. While the Mesozoic sediments are very similar to those encountered in the Paris Basin, the Cenozoic deposits are typical of the URG. The study area corresponds to the Pechelbronn sub-basin which which holds most of the petroleum reservoirs of northern Alsace (France), in the western central part of the URG.

In the Pechelbronn sub-basin, as elsewhere in the URG, it is noticed that the unconformity reaches the Middle Jurassic formations. Compared to the Paris Basin sedimentary successions, this questions why Upper Jurassic and Cretaceous formations are missing [START_REF] Schnaebele | Monographie géologique du champ pétrolifère de Pechelbronn[END_REF][START_REF] Ménillet | Notice explicative de la carte géologique de France (1/50000)[END_REF]. Especially for the Cretaceous sediments, it remains uncertain whether their absence is related to a sedimentary hiatus or a period of intense erosion as discussed by [START_REF] Ziegler | Collision related intra-plate compression deformations in Western and Central Europe[END_REF] or [START_REF] Blaise | Vertical and lateral changes in organic matter from the Mesozoic, eastern Paris Basin (France): Variability of sources and burial history[END_REF][START_REF] Blaise | Reconstruction of low temperature (<100°C) burial in sedimentary basins: A comparison of geothermometer in the intracontinental Paris Basin[END_REF]. This uncertainty is problematic to rebuild the burial history of Mesozoic sedimentary formations. Indeed, vitrinite reflectance values (Ro%) in well profiles present a sharp offset in Ro% values in the Jurassic series compared to the Tertiary [START_REF] Robert | Histoire géothermique et diagenèse organique[END_REF] suggesting that there may be two different geothermal histories to be considered.

Rock samples of Tertiary and Jurassic ages as well as petroleum samples were collected in the Pechelbronn area and analysed for their organic geochemistry. The geochemical analyses of the Jurassic outcrops show that the Mesozoic formations are immature or in the early oil window. If we consider that the pre-tertiary sedimentary column was not sufficiently buried before the Cretaceous, we can estimate a maximum paleo-burial and thus, evaluate the thickness of the eroded series. This estimation is made using 1D thermal models (PetroMod) from wells located in the Northern Alsace for which geological and Ro% data were published [START_REF] Robert | Histoire géothermique et diagenèse organique[END_REF].

The aim of this presentation will be to show evaluation of heat flux, oil generation and maximum paleo-burial in the Pechelbronn area by using molecular markers, vitrinite reflectance, kinetic properties of source-rocks and artificial maturation. The comparison between the values simulated by the 1D thermal basin models (PetroMod) and the measured values from the maturity markers allows calibration for these wells. Model calibration consists in estimating the thicknesses of the eroded series and heat flux values. Supplementary information are brought by comparing calculated thermal maturity of oils with geochemistry of collected oil samples. Special attention will be given to possible local heat anomalies linked

12-17 September 2021, Montpellier, France to hydrothermal fluid circulations, well known in the URG (Werner and Doebl, 1974;Lampe and Person, 2002;Baillieux, 2012). This work is funded by DEEPSURF project "Lorraine Université d'Excellence", ANR-15-IDEX-04-LUE.