EMIM 2018 ControlCenter

Online Program Overview Session: PS-23

To search for a specific ID please enter the hash sign followed by the ID number (e.g. #123).

Imaging Immune Disorders

Session chair: Leo Carlin - Glasgow, UK; Samantha Terry - London, UK
 
Shortcut: PS-23
Date: Friday, 23 March, 2018, 8:30 AM
Room: Lecture Room 03 | level -1
Session type: Parallel Session

Abstract

Click on an contribution preview the abstract content.

8:30 AM PS-23-1

Introductory talk by Vladimir Ponomarev - New York, USA

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

8:50 AM PS-23-2

Visualising regulatory T cell trafficking in pre-clinical humanised mouse transplantation models using SPECT/CT imaging (#106)

J. Jacob1, G. O. Fruhwirth3, R. I. Lechler1, L. A. Smyth1, 2, G. Lombardi1

1 King's College London, School of Immunology and Microbial Sciences, London, London, United Kingdom
2 University of East London, School of Health Sport and Bioscience, London, London, United Kingdom
3 King's College London, Rayne Institute, Imaging Sciences, London, London, United Kingdom

Introduction

Success of solid organ transplantation is hindered by attack from the recipient’s immune system which leads to rejection. Prolonged transplant survival can be achieved, in murine models, by favouring a regulatory environment via adoptive transfer of ex-vivo expanded regulatory T-cells (Tregs). Despite ongoing clinical trials using polyclonal Tregs, details of transferred Treg longevity and trafficking remain elusive. Here, we address whether longitudinal whole body nuclear imaging of radiolabelled human Tregs in vivo may help address the aforementioned points.

Methods

hNIS-GFP+ Tregs were generated and tested for phenotypic markers, suppressive capacity and ability to take up 99mTcO4-. hNIS-GFP+Tregs and autologous CD25- PBMCs were i/v injected into immunodeficient BRG (BALB/cRag2−/−gc−/−) or NSG (NOD/scid/IL-2Rg−/−) mice transplanted with human skin. Both BRG and NSG mice do not produce mature T or B cells, but NSG mice have an additional defect in innate immunity. Additionally, BRG mice were treated with or without anti-Gr1/Ly-6G antibody to deplete innate cells such as neutrophils. hNIS-GFP+ Tregs were tracked in vivo by SPECT/CT using 99mTcO4-  on days 0, 3, 8, 16, 25 and 40, and their location verified ex vivo by histology and flow cytometry. Ex vivo gamma-counting of transplant tissues confirmed in vivo imaging results.

Results/Discussion

Ex vivo expanded hNIS-GFP+ Tregs labelled with 99mTcO4 exhibited significant radioactive uptake in comparison to untransduced Tregs. Importantly, hNIS-GFP+ Tregs also retained their phenotype and suppressive ability following radioactive uptake (Fig.1). In vivo hNIS-GFP+ Tregs were observed as early as on day 3 post transfer in the skin graft and remained detectable until day 40 (Fig.2). Subsequent retrieval of hNIS-GFP+ Tregs from the graft confirmed Treg presence in the tissue, with a mean SUV of 0.61 at day 40 after injection (Fig.2C). Interestingly, we observed that the presence of mouse granulocytes impacted on Treg migration, as mice treated with anti-Gr1 antibody displayed delayed Treg presence in grafts, also seen with NSG mice (day 40).

Conclusions

This is the first study to show that SPECT/CT imaging using human hNIS-GFP reporter gene technology allows longitudinal Treg tracking in a transplant setting.

References

1. Regulatory T cells: tolerance induction in solid organ transplantation. Vaikunthanathan, T. et al. 2017. Clinical and experimental immunology. 189, 2, p197-210.

2. A whole-body dual-modality radionuclide optical strategy for preclinical imaging of metastasis and heterogeneous treatment response in different microenvironments. Fruhwirth, G. et al. 2014. Journal of Nuclear Medicine. 55, 4, p. 686-694

Acknowledgement

We acknowledge support from the Medical Research Council (MRC) for funding the PhD studentship, British Heart Foundation and Cancer Research UK.

Human Tregs can be transduced to express hNIS
hNIS-GFP Tregs can be visualised within the human skin graft of transplanted mice
Keywords: regulatory T cells, SPECT/CT imaging, cell tracking, transplantation, pre-clinical study
9:00 AM PS-23-3

Multimodal Assessment of Orbital Immune Cell Infiltration and Tissue Remodeling During Development of Graves’ Disease by 1H/19F MRI (#130)

U. Flögel1, A. Schlüter2, C. Jacoby1, S. Temme1, P. J. Banga2, A. Eckstein2, J. Schrader1, U. Berchner-Pfannschmid2

1 Heinrich Heine University, Experimental Cardiovascular Imaging, Düsseldorf, Germany
2 University Hospital Essen, Molecular Ophthalmology, Essen, Germany

Introduction

Graves’ disease is an autoimmune condition of the thyroid gland, leading to overproduction of thyroid hormones. A frequent complication is Graves’ orbitopathy (GO), where inflammation of the orbit associated with increased adipogenesis and deposition of hyaluronan results in detrimental remodeling of the orbital soft tissue (1,2). Since current diagnostics are limited in encompassing the complex phenotype of GO, the present study aimed to evaluate key molecular and cellular features of GO by simultaneous monitoring of alterations in morphology, inflammatory patterns, and tissue remodeling.

Methods

To this end, we utilized a murine model of GO induced by immunization with a human thyrotropin receptor (TSHR) A-subunit plasmid (3,4). Altogether 52 mice were used: 27 GOs and 25 controls immunized with b-galactasidose plasmid (Ctrl). From these, 17 GO and 12 Ctrl mice were subjected to multimodal MRI at 9.4T, while 23 mice only underwent histology. Beyond anatomical 1H MRI, we employed T2 mapping for visualization of edema, chemical exchange saturation transfer (CEST) for detection of hyaluronan, and 19F MRI for tracking of in situ labeled immune cells after intravenous injection of perfluorcarbons (PFCs). The full experimental protocol took around 60-90 min and was well tolerated by all mice.

Results/Discussion

1H/19F MRI demonstrated substantial infiltration of PFC-loaded immune cells in peri- and retro-orbital regions of GO mice (Fig. 1), while healthy Ctrls showed only minor 19F signals. In parallel, T2 mapping indicated onset of edema in peri-orbital tissue and adjacent ocular glands (P=0.038/0.017, Fig.1), which were associated with enhanced orbital CEST signals in GO mice (P=0.031). Concomitanty, a moderate expansion of retrobulbar fat (P=0.029) was apparent, but no signs for extraocular myopathy were detectable. 19F MRI-based visualization of orbital inflammation exhibited the highest significance level to discriminate between GO and Ctrl mice (P=0.006, Fig. 2), and showed the best correlation with the clinical score (P=0.0007). Infiltration patterns of inflammatory cells were corroborated by histology and examination of excised tissue after fixation: 3D high resolution 1H/19F MRI (isotropic voxel size 40 µm) confirmed the prevalent PFC deposition in the peri-orbital tissue of GO mice.

Conclusions

In the present study, we demonstrate that multimodal 1H/19F MRI permits a comprehensive analysis of several hallmarks of GO pathology with simultaneous assessment of orbital immune cell infiltration, development of edema, alterations in the extracellular matrix, and quantification of fat and muscle dimensions. In particular, 19F MRI allowed a sensitive demarcation of inflammatory foci in the orbit, even when other markers indicated only weak or no signs of tissue alterations.

References

  1. Bahn RS. Graves’ ophthalmopathy. N Engl J Med. 2010;362(8):726–738.
  2. Shan SJC, Douglas RS. The pathophysiology of thyroid eye disease. J Neuroophthalmol. 2014;34(2):177–185.
  3. Moshkelgosha S, So P-W, Deasy N, Diaz-Cano S, Banga JP. Cutting edge: retrobulbar inflammation, adipogenesis, and acute orbital congestion in a preclinical female mouse model of Graves’ orbitopathy induced by thyrotropin receptor plasmid in vivo electroporation. Endocrinology. 2013;154(9):3008–3015.
  4. Berchner-Pfannschmidt U, Moshkelgosha S, Diaz-Cano S, et al. Comparative assessment of female mouse model of Graves’ orbitopathy under different environments, accompanied by proinflammatory cytokine and T-cell responses to thyrotropin hormone receptor antigen. Endocrinology. 2016;157(4):1673–1682.

Acknowledgement

We gratefully thank Bodo Steckel (Heinrich Heine University), Mareike Horstmann (University of Duisburg-Essen, Germany) and Alexandra Brenzel (Imaging Center Essen IMCES, University Duisburg-Essen) for excellent technical assistance. The study was supported by Deutsche Forschungsgemeinschaft grants BE 3177/2-1 (UBP), FL 303/6-1 (UF), TE 1209/1-1 (ST), and by funding from the Stiftung Universitätsmedizin Essen (AE) and Interne Forschungsförderung Essen (AS).

Figure 1: 1H/19F MRI and T2 maps in GO and Ctrl mice
(A) 1H/19F MRI (left) reveals marked 19F signal intensity in peri-orbital regions of GO mice (top) while in corresponding T2 maps (right) only minor abnormalities compared to Ctrl mice (bottom) are visible. (B) Examples of merged 1H/19F MRIs with detectable 19F patterns in retro-orbital regions which was associated with an increase of T2 above 60 ms in the surrounding ocular gland (~57 ms in Ctrl)
Figure 2: Quantification of MRI data for GO and Ctrl mice
(A) 19F integral in vicinity of the orbit, (B) T2 of the Harderian gland, (C) CEST contrast in the orbit, and (D) fat volume surrounding the optic nerve; n = 17/12 for GO/Ctrl mice in A+D, 15/11 in C, and 11/9 in B; *P<0.05, **P<0.01; CI = confidence interval.
9:10 AM PS-23-4

Multicolor 19F-MRI for in vivo Imaging of immune cells activity (#400)

C. Chirizzi1, D. De Battista1, R. Furlan1, P. Metrangolo2, G. Comi1, F. Baldelli Bombelli2, L. Chaabane1

1 Ospedale San Raffaele, Institute of Experimental Neurology (INSPE), Milan, Italy, Italy
2 Politecnico di Milano, SupraBioNano Lab, Department of Chemistry, Materials, and Chemical Engineering “Giulio Natta”, Milan, Italy, Italy

Introduction

Inflammation is a dynamic process associated with several neurologic disorders and characterized by the involvement of different immune cells. MRI is a suitable imaging tool for in vivo investigation and demonstrated great potentials to image inflammation with the use of fluorine probes (1-4). In the present work, we extended 19F-MRI towards multicolor imaging using two different perfluorocarbons (PFCs) with two distinct resonance frequencies and a high fluorine payload. Furthermore, multicolor 19F-MRI was used to monitor in vivo the immune system activation in mice model of multiple sclerosis

Methods

Using two different PFCs, 19F-droplets were produced including fluorescent dyes for cytometric analysis (FCM). All in vivo experiments were performed at 7T in healthy and Experimental Autoimmune Encephalomyelitis (EAE) C57BL/6 mice. MRI at different fluorine frequencies of the PFCs and of hydrogen was acquired using a 3D-FSE sequence in all animals at different days post immunization (dpi). Before any signs of disease (4dpi), mice received a first dose of PFC and at disease onset (11dpi) the second PFC was administered. At disease peak (18dpi), animals received an additional dose of the first PFC. Mice were followed by MRI up to 22dpi. At the end of the study several organs were collected and processed for FCM to identify 19F-labeled cells.

Results/Discussion

Both 19F-droplets ( diameter = 180-200 nm) were clearly differentiated in vivo thanks to their single dominant resonance with 19 ppm of chemical shift allowing multicolor 19F-MRI without overlaps or artefacts. Fluorine signal was observed throughout the brain or localized in specific areas at the acute phase (Fig. 1a-b). Indeed, differences of 19F-signal were found in relation to the phases of EAE disease compared to healthy mice (Fig. 1c).

MRI observations were confirmed by FCM analysis of the brain where 10 to 20 % of cells were found positive to the second 19F-droplets in EAE mice while no cells were positive in healthy mice. These 19F-loaded cells were identified as monocytes, dendritic cells and microglia. In lymphoid organs, FCM analysis confirmed the presence of an inflammatory process with a high increase of monocytes compared to healthy mice (2 to 8 fold increase) and also a high 19F-uptake (2 to 5 fold increased versus healthy controls) with the second PFC at EAE onset

Conclusions

The present work demonstrates the potentiality of multicolor 19F-MRI with gain in sensitivity to track immune cells activation during different phase of disease progression in a model of multiple sclerosis. Interestingly, the proposed multicolor imaging method could be used for longitudinal in vivo investigations of cellular events which is of particular interest for the investigations of new therapies.

References

1. Temme S and al., 19F magnetic resonance imaging of endogenous macrophages in inflammation. WIREs Nanomed Nanobiotechnol; 2012.

2. Weise G et al., In vivo imaging of inflammation in the peripheral nervous system by 19F MRI. Experimental Neurology; 2011.

3. Caruthers SD et al., In vitro demonstration using 19F magnetic resonance to augment molecular imaging with paramagnetic perfluorocarbon nanoparticles at 1.5 Tesla. Invest Radiol; 2006.

4. Partlow KC et al. 19F magnetic resonance imaging for stem/progenitor cell tracking with multiple unique perfluorocarbon nanobeacons. FASEB J; 2007.

Acknowledgement

This study is supported by the Italian Multiple Sclerosis Foundation (FISM)

 

Figure 1. In vivo 19F-MRI.

Combined MR images acquired at different frequency (gray: 1H; green; PFC1; red: PFC2) in two EAE (a) and in a healthy mouse (b) at different phases of disease. (c). Comparison of signal from both fluorine tracers quantified in brain of healthy and EAE mice.

 

Keywords: 19F-multicolor MRI, fluorescence, in vivo imaging, perfluorocarbons
9:20 AM PS-23-5

Targeting activated synovial fibroblasts using photodynamic therapy in experimental arthritis (#274)

D. N. Dorst1, M. Rijpkema1, M. Buitinga2, M. Brom1, D. L. Bos1, A. Freimoser3, C. Klein3, B. Walgreen4, P. M. van der Kraan4, M. I. Koenders4, M. Gotthardt1

1 Radboud university medical center, Radiology and nuclear medicine, Nijmegen, Netherlands
2 KU Leuven, Leuven, Belgium
3 Roche Pharmaceutical Research and Early Development, Innovation center Zurich, Schlieren, Switzerland
4 Radboud umc, Experimental rheumatology, Nijmegen, Netherlands

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting synovial joints. In RA activated synovial fibroblasts (SF) are actively contributing to this inflammation. SF are characterized by the expression of Fibroblast Activation Protein (FAP). Here, we investigated the potential of photodynamic therapy (PDT) targeting FAP to selectively induce cell death in these cells. In targeted PDT, a light-sensitive molecule is delivered to a target cell and activated with light of a specific wavelength. This causes cell death through the production of reactive oxygen species (ROS).

Methods

Both ITC-DTPA and the photosensitizer IRDye700DX were conjugated to 28H1 (28H1-700DX). In vitro PDT assays were performed with 3T3 fibroblasts stably transfected with FAP. 3T3-FAP cells were incubated with 28H1-700DX or a control for 4 hours, and exposed to varying 690 nm light exposures. Subsequently, cell viability was measured using the CellTiter-Glo assay. For in vivo biodistribution, 5 days after onset of antigen-induced arthritis (AIA) C57Bl6 mice were injected with 28H1 labelled with 111-In, with or without 700DX. Additionally, micro-SPECT/CT and fluorescence imaging were performed. For PDT, arthritic mice were injected at day 5 of AIA with 28H1-700DX or PBS and exposed to 50 or 90 J/cm2 light 24 hours post injection. Joints were isolated at day 10 for histological analysis.

Results/Discussion

To assess PDT efficacy, we applied 13.7 J/cm2 light exposure to 3T3-FAP cells incubated with 6.67 pM 28H1-700DX, which significantly reduced cell viability (89.27%+/-2.48 compared to control (p<0.001)). No cell death was observed with the control 700DX-conjugate or with 3T3 fibroblasts not expressing FAP.

Conjugating the anti-FAP antibody to 700DX changed the in vivo biodistribution of the antibody, with a higher accumulation in the liver (27.06±0.95 %ID/g vs. 6.08±0.42 %ID/g with control (p<0.001)) and lower blood levels (5.32±0.36 %ID/g vs. 12.72±0.80 %ID/g with control (p<0.001)). Accumulation in the arthritic joints was not significantly different. The fluorescent signal still visible in the inflamed knee joint 24h post injection indicates that there is intact tracer accumulation, as previous experiments indicated that the fluorescent signal and the ability of 700DX to produce ROS are linearly correlated. Histological analysis of the PDT-treated mouse knee joints is ongoing.

Conclusions

We have demonstrated fibroblast-specific cell death using 700DX-conjugated 28H1 PDT, indicating FAP-based targeted PDT as a promising new tool in treating RA. Furthermore, we demonstrated that adding 700DX results in faster liver clearance of the antibody conjugate, but does not affect uptake in the inflamed knee joint. Visualization of fluorescent signal from the 28H1-700DX construct indicates that  the photosensitizer is intact and capable of producing ROS at the site of inflammation. Future research will further elucidate the applicability of our conjugate for PDT in animal models of RA.

Increased uptake of the 28H1-700DX tracer in an inflamed joint visualized by fluorescent signal.
Fluorescence measured at 700nm using the IVIS® Spectrum in vivo imaging system in paws of a mouse with antigen induced arthritis 24 hours post injection of the 28H1-700DX construct. Note the higher fluorescent signal in the inflamed knee joint (left) compared to the control knee joint (right).
Keywords: Photodynamic therapy, Rheumatoid arthritis, Experimental arthritis
9:30 AM PS-23-6

Molecular imaging of elastin and collagen deposition in renal fibrosis (#235)

M. Baues1, Q. Sun2, 3, B. Klinkhammer2, 3, J. Ehling1, 2, P. Boor2, 3, F. Kiessling1, T. Lammers1

1 Uniclinic RWTH Aachen, Institute for Experimental Molecular Imaging, Aachen, Germany
2 Uniclinic RWTH Aachen, Institute of Pathology, Aachen, Germany
3 Uniclinic RWTH Aachen, Department of Nephrology and Immunology, Aachen, Germany

Introduction

Millions of patients suffer from chronical kidney disease (CKD). The best predictor for CKD progression is fibrosis assessment. Biopsies have remained the gold standard, even if highly invasive and moderately informative. Fibrosis refers to deposition of extracellular matrix (ECM) components, such as elastin and collagen. Fibrogenesis is characterized by disease state-specific ECM compositions. Based on these notions, we employed the molecular imaging agents: ESMA [1] and CNA35 [2], which specifically target elastin and collagen, for the diagnosis and staging of renal fibrosis (Fig.1A).

Methods

The agents were evaluated using magnetic resonance imaging (MRI) and hybrid computed tomography - fluorescence molecular tomography (CT-FMT). This was done in different mouse models: unilateral ureteral obstruction (UUO), ischemia/reperfusion injury (I/R) and adenine-containing diet (Fig.1B [3]). Findings were verified by laser ablation inductively coupled mass spectrometry (LA-ICP-MS) and two-photon laser scanning microscopy (TPLSM).

Results/Discussion

Western blot and immunohistochemistry confirmed gradual deposition of elastin during disease progression (Fig.2A). For the elastin-specific contrast agent ESMA, normalized signal intensities in T1-weighted MRI as well as T1 relaxometry acquisition with echo time analysis, revealed significant differences in fibrotic vs. healthy kidneys 24 h after i.v. injection, which was clearly stronger than the difference observed for unspecific Gd-DTPA (Fig.2B). The metal quantification via LA-ICP-MS reflected the MRI signal intensities and visualized highest Gd concentrations in the fibrotic kidneys injected with ESMA (Fig. 2C). For the Cy7- and Alexa488-labeled collagen-specific agent CNA35 CT-FMT imaging revealed high accumulation in fibrotic (F) kidneys while only moderate amounts accumulate in the contralateral healthy (H) kidney (Fig.2D-E). TPLSM confirmed these findings, showing highly specific binding of CNA35 to perivascular collagen fibers at 4 and 24 h after i.v. injection (Fig.2F).

Conclusions

We established probes and protocols for molecular imaging of CKD. Our findings lay the basis for the establishment of elastin- and collagen-based imaging biomarkers that hold potential for non-invasive, quantitative and longitudinal analysis of renal fibrosis.

References

[1] Makowski M et al. Nat Med 2011, 17:383-388

[2] Sanders H et al. Chem Commun 2011, 47:1503-1505

[3] Ehling J et al. J Am Soc Nephrol 2016, 27:520-532

Acknowledgement

Supported by ERC (StG309495-NeoNaNo), DFG (SFB/TRR57) and START (124/14, 152/12).

Figure 1
Imaging biomarkers and mouse models for diagnosis and staging of kidney fibrosis.
Figure 2
Molecular imaging of elastin with ESMA and collagen with CNA35 in kidney fibrosis.
Keywords: Imaging biomarker, ECM, Non-invasive, MRI, CT-FMT, kidney fibrosis
9:40 AM PS-23-7

Tracking the Delivery and Assessment of the efficacy of Fluorescent Glucocorticoid Hybrid Nanoparticles in Experimental Mouse Models of Inflammation (#413)

J. Napp1, 2, 3, A. M. Markus1, J. G. Heck4, C. Dullin2, C. Feldmann4, F. Alves1, 2, 3

1 MPI of Experimental Medicine, Translational Molecular Imaging Group, Göttingen, Germany
2 University Medical Centre Goettingen (UMG), Institute of Diagnostic and Interventional Radiology, Göttingen, Germany
3 University Medical Centre Goettingen (UMG), Clinic of Hematology and Medical Oncology, Göttingen, Germany
4 Karlsruhe Institute of Technology, Institute of Inorganic Chemistry, Karlsruhe, Germany

Introduction

Effective treatment of a disease obviously depends on the delivery of the therapeutic agents to the site of action. Therefore, the aim of the study was to evaluate the monitoring of the in vitro and in vivo distribution, delivery and uptake and the assessment of the efficacy of inorganic-organic hybrid nanoparticles (IOH-NPs) composed of an anti-inflammatory glucocorticoid, betamethasonphosphate (BMP) and a near-infrared fluorescent (NIRF) dye DY-647 (BMP-IOH-NPs)1.

Methods

BMP-IOH-NPs uptake by MH-S macrophages was analyzed with NIRF- and electron-microscopy. Lipopolysaccharide (LPS)-stimulated cells were treated for 48h with BMP-IOH-NPs (1x10-5-1x10-9 M), BMP or dexamethasone (DM) and drug efficacy was assessed by measurements of interleukin 6 (IL-6). Mice with Zymosan-A- induced paw inflammation were intraperitoneally treated with BMP-IOH-NPs (10 mg/kg) and mice with OVA-induced allergic airway inflammation (AAI)2 were treated intranasally with BMP-IOH-NPs, BMP or DM (2.5 mg/kg). Delivery was monitored by in vivo optical imaging (OI). Efficacy was assessed in vivo via paw volume measurement with µCT and ex vivo via paw weight quantification or in the AAI model by cell counts in bronchio-alveolar lavage (BAL) fluid histology and x-ray based lung-function³.

Results/Discussion

In vitro, an uptake of BMP-IOH-NPs by MH-S cells was observed during the first 10 min of incubation, with NPs load increasing over time. The anti-inflammatory effect of BMP-IOH-NPs on MH-S cells was dose dependent and comparable to that of DM and BMP (Fig. 1).

In vivo, the Zymosan-A injections induced inflammatory paw swelling (222 mm³ and 0.229 g in controls) was significantly reduced in mice treated with BMP-IOH-NPs (180 mm³ and 0.186 g) (Fig. 2A). OI showed accumulation of BMP-IOH-NPs within the lung of AAI mice 1h after instillation, detectable for at least 4h in vivo (Fig. 2B). BMP-IOH-NPs were preferentially taken up by peribronchial and alveolar M2 macrophages which were CD68+CD11c+ECF-L+MHCII- and podoplanin-proS-PClow. Treatment of AAI mice with BMP-IOH-NPs, but not with BMP, significantly reduced the number of eosinophils in BALs and immune cell infiltration in lungs, with an efficacy higher than the one of the golden standards, DM and BMP (Fig. 2C).

Conclusions

We show that glucocorticoids such as BMP applied in form of IOH-NPs allow efficient treatment of inflammatory disease by release of the active drug and simultaneous non-invasive monitoring of the delivery of the NPs by OI.

References

1Heck et al., J Am Chem Soc. 2015; 137:7329-36

2Markus et al., ACS Nano. 2015; 9:11642-57

3Dullin et al., J Synchrotron Radiat. 2015; 22:1106-11

Acknowledgement

Technical assistance: B. Heidrich, S. Garbode, B. Jeep, S. Wolfgramm

Fig. 1: In vitro uptake and efficacy of BMP-IOH NPs.
Fig. 2: Assessment of the in vivo efficacy and monitoring the distribution of BMP-IOH-NPs
9:50 AM PS-23-8

Evaluation of different treatments effectiveness preventing the development of pulmonary fibrosis with Micro-CT imaging. (#448)

J. Sadoine1, J. Avouac2, 3, M. Elhai2, 3, A. Cauvet2, S. Pezet2, Y. Allanore2, 3

1 Université Paris Descartes, EA2496 & Plateforme Imageries du vivant, Montrouge, France
2 Université Paris Descartes, INSERM U1016 CNRS UMR8104, Paris, France
3 Université Paris Descartes, Service de Rhumatologie A, Paris, France

Introduction

The X-ray micro-tomography (micro-CT) allows acquisition of structural images in order to obtain, with high resolutions, spatial representations of scanned (bio)-materials. Recent technical advances provide fast image acquisition allowing a long-term follow up of pathologies in small animal models. This technique is very sensitive and allows the evaluation of lung pathology and particularly lung fibrosis. Indeed, in the present study we describe the development of an image analysis method to explore the effect of three treatments against lung fibrosis associated with preclinical X-ray imaging.

Methods

We work with the Fra-2 mouse model, which is characterized by the spontaneous development of a progressive non-specific interstitial pneumonia. Tridimensional images were acquired with Micro-CT. Mice were anesthetized and respiratory frequency was recorded during the acquisition and only the inhalation was reconstructed. Fibrosing alveolitis was evaluated using micro-CT 2 d before mice were sacrified. Means of lung density of both groups were determined by evaluating all CT scans acquired from the apices to the bases of the lungs. Furthermore, the volume of functional lung parenchyma corresponding to functional respiratory capacity was drawn manually, excluding fibrotic area and vessels. The efficacy of 3 treatments (OX40L, IVA337 & MMP10) was evaluated on these parameters.

Results/Discussion

In a first study, the efficacy of a targeted therapy against OX40L was explored. Micro-CT revealed higher lung density consistent with fibrosing alveolitis in Fra-2 mice treated with control mAb than in C57/BL6 mice (P = 0.007); this lung density was decreased significantly in Fra2 mice treated with the anti-OX40L mAb (P = 0.004). Fra-2 mice had a functional residual capacity equal to 44.7% of lung volume versus 77.4% in control mice (P < 0.001). Fra-2 mice receiving anti-OX40L mAb had a functional residual capacity of 71.4% of lung volume. After, we tested the efficacy of IVA337 in the Fra-2 mouse model, Fra-2 mice treated with IVA337 100 mg/kg displayed a significant 21% decrease in lung density as compared with Fra-2 mice receiving the vehicle (p<0.05). Consistent with this finding, functional residual capacity increased significantly by 30% in mice treated with IVA337 100 mg/kg (p<0.05). Moreover, MMP10 inhibition failed to improve significantly fibrosing alveolitis in Fra-2 model.

Conclusions

Using systemic sclerosis (SSc) as a prototypic disease, we report compelling evidence that blockade of OX40L is a promising strategy for the treatment of inflammation-driven fibrosis. We demonstrate that treatment with 100 mg/kg IVA337 prevents also lung fibrosis in the Fra-2 mouse model. The present work shows how X-ray micro-CT imaging and image segmentation/quantification developped for the 3 differents study projects are suitable technique and method to quantify specific parameters (as tissue density or functionnal residual capacity) associated to lung pathologies as pulmonary fibrosis.

References

[1] Elhai M, Avouac J, Hoffmann-Vold AM, Ruzehaji N, Amiar O, Ruiz B, Brahiti H, Ponsoye M, Fréchet M, Burgevin A, Pezet S, Sadoine J, Guilbert T, Nicco C, Akiba H, Heissmeyer V, Subramaniam A, Resnick R, Molberg Ø, Kahan A, Chiocchia G, Allanore Y.OX40L blockade protects against inflammation-driven fibrosis.  Proc Natl Acad Sci U S A. 2016 Jul 5;113(27):E3901-10.

[2] Avouac J, Konstantinova I, Guignabert C, Pezet S, Sadoine J, Guilbert T, Cauvet A, Tu L, Luccarini JM, Junien JL, Broqua P, Allanore Y. Pan-PPAR agonist IVA337 is effective in experimental lung fibrosis and pulmonary hypertension.  Ann Rheum Dis. 2017 Aug 11. pii: annrheumdis-2016-210821. doi: 10.1136/annrheumdis-2016-210821.

[3] Avouac J, Guignabert C, Hoffmann-Vold AM, Ruiz B, Dorfmuller P, Pezet S, Amar O, Tu L, Van Wassenhove J, Sadoine J, Launay D, Elhai M, Cauvet A, Subramaniam A, Resnick R, Hachulla E, Molberg Ø, Kahan A, Humbert M, Allanore Y. Stromelysin-2 (MMP-10), a novel mediator of vascular remodeling underlying pulmonary hypertension associated with systemic sclerosis.  Arthritis Rheumatol. 2017 Aug 13. doi: 10.1002/art.40229.

Acknowledgement

We thank the following individuals for excellent technical assistance: ..... and Prof. Catherine Chaussain & Dr. Lotfi Slimani (Dental School of the Paris Descartes University, EA 2496 & Life Imaging Facility of Paris Descartes University (Plateforme Imageries du Vivant - PIV)).

Inhibition of OX40L prevents the development of fibrosing alveolitis: CT-scan data
A, Fibrosing alveolitis was observed in Fra-2 mice receiving control IgG. B, Representative images of functional residual capacity (in blue) in different mice; bronchi are in white. C, Increasing lung density in Fra-2 mice treated with control IgG compared with Fra-2 mice treated with anti-OX40L mAb or Control mice. D, Residual lung volume.**P < 0.01; ***P < 0.001; two-sided Mann–Whitney test.
IVA-337 100 mg/kg prevents lung fibrosis in Fra-2 transgenic mice: Evaluation by CT-scan.

A, Representative pictures of micro-computed tomography. B, Decreased lung density at micro-computed tomography in Fra-2 transgenic mice treated with IVA337 100 mg/kg compare to vehicle-treated mice. C, Representative pictures of functional residual capacity in different mice in blue. D, Reduced residual lung volume. Values the mean ± SEM. Statistics: One way ANOVA test. * p<0.05

Keywords: Micro-CT, Systemic sclerosis, Fibrosis, Image analysis