Involvement of an IgE/Mast cell/B cell amplification loop in abdominal aortic aneurysm progression

Aims IgE type immunoglobulins and their specific effector cells, mast cells (MCs), are associated with abdominal aortic aneurysm (AAA) progression. In parallel, immunoglobulin-producing B cells, organised in tertiary lymphoid organs (TLOs) within the aortic wall, have also been linked to aneurysmal progression. We aimed at investigating the potential role and mechanism linking local MCs, TLO B cells, and IgE production in aneurysmal progression. Methods and Results Through histological assays conducted on human surgical samples from AAA patients, we uncovered that activated MCs were enriched at sites of unhealed haematomas, due to subclinical aortic wall fissuring, in close proximity to adventitial IgE+ TLO B cells. Remarkably, in vitro the IgEs deriving from these samples enhanced MC production of IL-4, a cytokine which favors IgE class-switching and production by B cells. Finally, the role of MCs in aneurysmal progression was further analysed in vivo in ApoE-/- mice subjected to angiotensin II infusion aneurysm model, through MC-specific depletion after the establishment of dissecting aneurysms. MC-specific depletion improved intramural haematoma healing and reduced aneurysmal progression. Conclusions Our data suggest that MC located close to aortic wall fissures are activated by adventitial TLO B cell-produced IgEs and participate to their own activation by providing support for further IgE synthesis through IL-4 production. By preventing prompt repair of aortic subclinical fissures, such a runaway MC activation loop could precipitate aneurysmal progression, suggesting that MC-targeting treatments may represent an interesting adjunctive therapy for reducing AAA progression.


Introduction
Arteries are subjected to recurrent mechanical insults which rise from the luminal side of 2 vessels (1). Vessels have intrinsic capacities to ensure prompt healing of local injuries and are 3 assisted in this task by resident and recruited inflammatory cells (2). However, the persistence 4 of vascular inflammation can eventually amplify the arterial damage and lead to severe and life-5 threatening conditions including coronary artery disease, strokes or abdominal aortic 6 aneurysms (AAAs) (3). Promoting arterial healing by targeting inflammation thus constitutes a 7 major challenge in modern medicine. 8 In this study, we focused on AAA progression. Monitoring AAA enlargement is problematic 9 due to the discontinuous, so-called 'staccato' growth where month-lasting no-growth/healing 10 periods can be succeeded by a sudden enlargement and again a no-growth/healing period (4). 11 So far, no pharmacological treatments have been identified to reduce or stop AAA expansion 12 and subsequent arterial rupture, which causes up to 200,000 deaths worldwide each year (5). 13 The only therapeutic option when the aneurysm diameter exceeds a certain value (55mm in 14 men, 50mm in women) is aorta surgery. Therefore, deciphering the molecular pathways 15 involved in AAA healing and/or progression is essential to set up pharmacological alternatives. 16 Chronic immune responses involve adaptive and innate immunity and their relationship. In 17 particular, we and others have observed that chronic immune stimulation in AAAs leads to local 18 tertiary lymphoid organ (TLO) development within the adventitia (6-9). TLOs contain germinal 19 centre B cells corresponding to B cells undergoing differentiation into plasma cells (6). We 20 have reported that TLO development is associated with increased levels of antibodies in the 21 adventitia of human AAAs, in particular IgEs (6), which are also involved in chronic 22 inflammation. Notably, increase in IgE blood concentration correlates with the progression of 23 then 40 cycles of 3 steps: denaturation at 95°C 15 seconds, annealing at 57°C for 15 seconds, 1 and a final extension at 72°C for 30 seconds. The data were analysed using the 2 -DDCt formulas: 2 the Ct values of IL-4 were normalised to the average Ct values of RPS18 (forward RPS18: 3 GCGGCGGAAAATAGCCTTTG; reverse RPS18: GATCACACGTTCCACCTCATC) and 4 ACTB (forward ACTB: TCCCTGGAGAAGAGCTACG; reverse ACTB: 5 TTTCGTGGATGCCACAGGAC) and non-treated MCs were used as reference. 6 Ang-II abdominal aortic pseudoaneurysms in mice 7 28-week-old ApoE-RMB males were used for the experiments, as males are more susceptible 8 to develop AAAs after Angiotensin II ( AngII) infusion (25). AngII (#A9525, 1 9 mg/kg/day, Sigma-Aldrich, St Louis, Missouri) was continuously infused into the experimental 10 mice via osmotic mini-pumps (Model 2004, Alzet, Charles River Laboratories) that were 11 surgically implanted subcutaneously in the interscapular region under anaesthesia induced by 12 intraperitoneal injection of ketamine (100 mg/kg) and xylazine (20 mg/kg). Surgery was 13 followed by a buprenorphine intraperitoneal injection (0.1 mg/kg) for analgesia. Three mice 14 (8%) died within the first 10 days. Fourteen days after inducing AAAs through AngII infusion 15 (26), mice received two intraperitoneal injection of DT (1µg/ injection, n=18) at a one-day 16 interval (or PBS in the control group, n=17) in order to deplete MCs. Randomization was 17 achieved by having mice from each experimental group in each cage. Mice were sacrificed at 18 day 28 by intracardiac exsanguination under overdose of anaesthesia (intraperitoneal injection 19 of 150 mg/kg ketamine and 30 mg/kg xylazine). Before the exsanguination, a cell suspension 20 from the peritoneum was obtained by peritoneal lavage with 5 mL of ice-cold PBS. Blood was 21 withdrawn from the right ventricle of the heart and collected in EDTA tubes for blood cell 22 analysis. The heart and aorta were dissected, photographed and mounted on cryomolds for 23 further histomorphological analysis based on cryosections. The experiment was repeated twice 24 with similar results. Pooled number of mice from both experiments who survived and developed aneurysms in each group are summarized in Table S3. DT injection had no effect on 1 blood basophils, peritoneal MCs, and aortic tissue MCs in control ApoE -/mice (data not 2 shown). 3 Histology on mouse tissues 4 MC depletion was analysed on cryosections of mouse aortic roots stained with Toluidine Blue. 5 Images were captured on a Zeiss Axio Observer Z7 inverted microscope, and MCs were 6 counted manually. A detailed blind analysis of pseudoaneurysms was performed for mouse 7 abdominal aortas displaying macroscopic evidence of the occurrence of a pseudeoaneurysm. 8 Cross-cryosections (10 µm) of the aortic segments taken at different levels of the 9 pseudoaneurysms (every 300 µm) were stained for collagen with picrosirius red. Images were 10 acquired under polarised light. The size of the different layers (media, haematoma, adventitial 11 fibrous cap) and the extent of collagen deposition were quantitatively assessed using Image J. 12

Image acquisition
13 Images were digitally captured using an AxioObserver epi-fluorescent microscope equipped 14 with a Colibri 7 LED generator (Zeiss) and an ApoTome system and running Zen Software 15 (Zeiss). Macroscopic images of the human samples were acquired using the NanoZoomer 16 Digital slide scanner (Hamamatsu Photonics). 17 Tissue infiltrated IgE were enriched in aneurysmal samples and soluble IgE plasma levels were 4 elevated in AAA patients compared to control non-aneurysmal aortas (NAAs, Fig S1). In 5 parallel, TLOs were found in a majority of AAA samples (20 out of 25, Fig 1A) whereas they 6 were absent in all NAA, in agreement with previous findings (6-9). In particular, adventitial 7 TLOs were consistently found in samples from patients with larger aneurysms (Fig 1B), 8 suggesting their higher frequency in these patients. In these adventitial TLOs, the proportion of 9 germinal centre B cells was dramatically increased (Fig S2), suggesting that adventitial TLO B 10 cells in large aneurysms are engaged into an Ab-producing program from which diverse Ig 11 isotypes could emerge. Interestingly, we observed a strong staining for IgEs within the TLO 12 "light zone" (where the centrocytes, i.e. B cells differentiating in immunoglobulins-producing 13 cells, are located (28); Fig 1A) in 45% (n=9) of the samples. Altogether these results suggest 14 that germinal centre B cells within adventitial TLOs situated within large AAAs may actively 15 produce IgE immunoglobulins. 16 Intriguingly, combined histological analysis of Carstair's, Perl's + DAB and Orcein staining 17 revealed the presence of recent or past wall fissuring with haematoma formation in the media 18 in 92% AAA samples ( Fig 1C, Fig S3, and Table 1). Indeed, 84% of total AAA tissues (n=21) 19 presented an accumulation of red blood cells (Fig 1C), reflecting the recent occurrence of 20 intramural haematomas. Of note, the diameter of the aneurysmal samples was significantly 21 larger than 3 cm, implying a greater tension and mechanical stress, than the one of the non-22 aneurysmal aortas (diameter < 3 cm) (5) and an attentive evaluation of pre-surgery tomography 23 angiograms consistently revealed the presence of at least one detectable aneurysmal wall macroscopic fissure, with radiologic contrast penetrating via blood disruptions from the aortic 1 lumen to the aortic wall through the intraluminal thrombus, in 78% of patients with intramural 2 haematomas ( Fig 1D and Fig S3D). In some samples, we were able to observe the entry site of 3 blood from the lumen on AAA histological sections, suggesting that the fissures leading to 4 intramural haematomas were provoked by tears (micro-fissures) initiated from the aorta lumen 5 ( Fig S4A-B and S4D-E). Unhealed intramural haematomas could also be evidenced as large 6 areas of ferrous iron deposits next to modest red blood cell accumulation in the media (Fig S3B)  7 in 8% (n=2) of samples. Importantly, IgE+ TLOs were frequently localized near these 8 intramural haematomas (Fig 2A-C), suggesting that the maturation of B cells towards IgE-9 producing cells within adventitial TLOs and the occurrence of micro-fissures are linked. Given that MCs displayed an activated phenotype only in the adventitia of AAA patients (Fig  6   2), we assessed the putative presence of MC activation triggers in the conditioned medium 7 prepared from the adventitia of AAA as compared to control NAA tissues. We observed that 8 conditioned medium from AAA adventitias induced more surface expression of the 9 degranulating marker CD63 and more IL-4 mRNA transcription than conditioned medium from 10 NAA adventitia in cultured ROSA human MCs (Fig 3A-B). This is important because the 11 cytokine IL-4 is required to carry out an IgE antibody class switch recombination and hence 12 allow the production of IgE by B cells (29). Therefore, these results point at an unforeseen 13 function of MCs in AAAs, whereby MCs could orientate the local adaptive immune response 14 generated in adventitial TLOs toward IgE production. In turn, the binding of adventitial IgE on 15 their high affinity receptor on MCs could be responsible for triggering MC activation. To 16 directly assess this hypothesis, we pre-incubated the conditioned medium with anti-IgE 17 DARPin ® protein bi53_79 (5 µM) before adding it to MCs (Fig 3C-D). DARPin ® protein 18 bi53_79 is a soluble molecule that specifically binds to the IgE-Fc portion thereby preventing 19 IgE binding to FceRI (24). Whereas treatment of conditioned medium with bi53_79 did not 20 prevent MC degranulation (Fig 3C), it decreased IL-4 mRNA production compared to untreated 21 conditioned medium from AAA tissues (Fig 3D). IgEs contained in the conditioned medium 22 from AAA adventitias therefore emerge as instrumental in promoting MC IL-4 production.
To assess if IL-4 production, MCs, and IgEs are also linked in vivo, we measured IgE, tryptase, 1 and IL-4 quantities in the conditioned medium from AAA adventitias. We found a statistically 2 significant positive correlation between the concentration of IgEs and IL-4 (p<0.001), as well 3 as between the concentration of IL-4 and tryptase (p<0.05, Fig 3E-F). No such correlations 4 were observed in the plasma of paired patients (Fig S6). These observations support the 5 existence of a local amplification loop in the adventitia of AAAs involving IL-4 producing MCs 6 and IgE-producing B cells. 7 8

9
Previous studies have identified MCs as directly involved in provoking arterial damage (13, 14, 10 19). Consistently, the presence of medial/adventitial MCs with an activated phenotype was 11 associated to micro-fissures in our human AAA samples, suggesting that MCs could play a role 12 in the aneurysmal remodelling of the aortic wall, upon the occurrence of tissue fissuring. 13 Therefore, we asked whether specific depletion of MCs could prevent the aneurysmal 14 remodelling in ApoE -/mice subjected to chronic infusion of Angiotensin II (AngII), a well-15 known mouse model of aortic dissection eventually followed by aneurysmal progression (26). 16 In this setting, pseudoaneurysm of the abdominal aorta starts within 10 days upon the formation 17 of large intramural haematomas due to the occurrence of multiple medial tears originating from 18 the lumen, at the origin of the main side branches (30). In order to conditionally induce specific 19 MC depletion by DT injection after the occurrence of the aortic fissuring, we crossed ApoE -/-20 mice with RMB mice (ApoE-RMB mice) (21). ApoE-RMB mice were subjected to infusion 21 with AngII for 28 days. We injected DT (or PBS as a control) 14 days after the beginning of 22 AngII infusion. Thus, MCs were depleted after the occurrence of the dissection in this model 23 (26), allowing to evaluate the effect of their depletion on the remodelling of the dissected aortas 24 and their potential effect on aneurysmal progression 14 days later (Fig 4A).
Survival was similar in the two groups (Table S3). As anticipated, the proportion of mice 1 presenting an intramural haematoma at day 28 was equivalent in DT-treated mice (44%, n= 2 8/18) and in the control group (47%, n=7/17). DT injection almost completely depleted MCs 3 from the aortic tissue (Fig 4B-C) and the peritoneal cavity (Fig S7). It should be noted that in 4 RMB mice, DT induces transient depletion of both MCs and basophils. While basophils fully 5 repopulate the bloodstream within 12 days, the repopulation of peripheral tissues such as skin 6 or peritoneum by MCs is much slower (21, 31). As a result, two weeks after DT administration 7 (Day 28 of AngII infusion), MCs were still completely absent from the peritoneal cavity, 8 whereas the basophil population had fully replenished the blood of ApoE-RMB mice (Fig S7). 9 DT injection reduced the expansion of the pseudoaneurysm (Fig 4D-F), comprising the size of 10 the intramural haematoma and of the perivascular adventitial cuff (Fig S8A-C). Furthermore, 11 on the cross-sections of the dissected aorta segments stained with picrosirius red we observed 12 that the aortic wall of the pseudoaneurysm of DT-treated mice presented an increased density 13 of collagen compared to control mice (Fig 4E and 4G), especially in the adventitial fibrous cuff 14 ( Fig S8D). Our data therefore suggest that MC depletion improves the adventitia fibrotic purified IgEs by an anti-IgE antibody for activation (23). However, we did not induce such 23 cross-linking with the conditioned medium. The activation of MCs under these experimental conditions suggests that the IgEs present in the conditioned medium were likely cross-linked 1 by their cognate antigens. In contrast, our data indicate that blood IgEs (not shown) failed to 2 activate MCs, suggesting that they were not circulating as immune complexes. This strongly 3 suggests that the antigen recognized by IgEs may be specifically present in the aneurysmal 4 aorta. Additionally, locally produced IgEs may contribute to AAA progression by promoting 5 the senescence of smooth muscle cells (35). 6 Whereas MC IL-4 production was dependent on IgEs present in the conditioned medium from 7 aneurysmal aorta, degranulation was not. This indicates that besides IgEs, other molecules from 8 the adventitia stimulate MCs. Indeed, other classes of immunoglobulins, which are also 9 enriched in aneurysmal aortas (6), cytokines, DAMP-containing molecules and inflammatory 10 mediators released by the injured vascular stroma could also trigger MC degranulation. Further 11 studies focusing on the relationship between IgEs and MC activation could lead to novel 12 therapeutic strategies to reduce chronic inflammatory disorders. This trial in which the major end-point was the change in aortic diameter as assessed by ultra-16 sound imaging, failed to report a significant improvement in the patients receiving the MC 17 inhibitor. Although this study was elegantly designed and performed, it did not however bring 18 a definitive conclusion regarding MC involvement in AAA pathophysiology because i/ the 19 pemirolast treatment failed to decrease the plasma tryptase levels, indicating a critical dosing 20 issue, ii/ the study interval might have been too short, and iii/ contrary to our study, the effect 21 that MC inhibition might have exerted on tissue composition could not be studied in this clinical 22 study. Hence, in light of our results, we believe it would be interesting to re-evaluate the effect 23 of perimolast (and/or other MC-inhibiting drugs) on AAA growth and rupture rate by targeting 24 patients presenting elevated levels of IgEs and focusing on adventitial remodelling. Inhibiting other key steps of the amplification loop, for instance by using anti-IgE antibodies, could be 1 another promising therapeutic strategy for such patients. 2 The present study has several limitations that should be acknowledged.   Values are mean +/-SEM or %. Ancient intramural haematomas are characterised by ferrous 3 iron deposits with few red blood cells in the media (Supplemental Figure 3B) and recent 4 intramural haematomas are characterized by abundant red blood cells in the media ( Figure 1C). 5 1   Table S1: Patients'characteristics. 2 Table S2: NAA healthy donors'characteristics. 3 Table S3: ApoE-RMB survival and pseudoaneurysm occurrence.