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Background/Introduction Bicuspid aortic valve (BAV) is associated with ascending aorta aneurysms and dissections. Presently, genetic factors and pathological flow patterns are considered responsible for aneurysm formation in BAV. Despite, indication for preventive surgery is presently only defined by vessel diameter which is poor marker for dissection, as it does not take other processes responsible for the vulnerability of the aorta into account. Purpose Inflammation is not considered a player in BAV aortopathy. We introduce a quantitative immunohistochemistry (IHC) approach to sensitively look at the potential role of both the innate and adaptive immune system in BAV aortopathy. Methods Dilated (n=8), non-dilated (n=14) and dissected (n=4) BAV ascending aortas were collected during surgery or from post-mortem donors. Median time from symptoms to surgery for dissections was just over 4 hours. Tissue was stained with a novel 8-colour IHC technique allowing for simultaneous visualization of 6 markers per slide, completed with DAPI nuclear counter-stain and elastin fiber autofluorescence. One panel focused on the adaptive immune system (identifying B cells and classic dendritic cells type 2 (cDC2s) and phenotyping T cells), and the other on the innate immune system (assessing macrophage polarization and neutrophil extravasation). All cells were identified and comprehensively phenotyped using automated quantitative analysis. Results Aneurysm formation was associated with an organized and consistent increase of lymphocytes in the adventitia. B cell follicles and helper T cell expansion were identified, suggestive of a targeted adaptive immune response (Fig. 1a). Only dissected aortas showed a statistically significant increase of helper T (p=0.3) and cDC2s (p=0.3) in the media, when compared to non-dilated and dilated samples (Fig. 1b). The short time between dissection symptoms and surgery suggests these cells were present before the dissection occurred. Furthermore, aneurysms and dissections are associated with a shift in macrophage phenotype to the more aggressive M1-like subset. In summary, we found that a progression of aggressive immune cells in the adventitia and media was correlated to a progression in disease state; from normal to dilated to dissected. Conclusions Aorta dilatation in patients with BAV is associated with an expansion of B and helper T cells in the adventitial compartment without changes in the media. This result might indicate an antigen-driven adaptive immune response. Only dissections show an increase in helper T cells and cDC2s in the media, together with polarization of macrophages to a more M1-like phenotype. We hypothesize that antigen-specific helper T cells expand in the adventitia, migrate to the media, and then potentiate macrophages which can eventually lead to tissue degeneration. These associations could shine light on the final step in the deterioration of the aorta towards a dissection. Figure 1. Microscopy results Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): TTW-NWO open technology grant (STW-14716), ERC-2014-StG-336454-CoNQUeST

Background Chronic Q fever (QCF) is the persistence of C. Burnettii after an acute infection. Atheromas in the abdominal aorta are a preferential location of this bacterial persistence and may result in accelerated abdominal aorta aneurysm (AAA) formation or even rupture. CQF is characterized by a poor response to antibiotic therapy with high mortality. The localization of the bacteria in the acidic lysosomes is thought to be the main cause of poor antibiotic efficacy. Purpose A potent immune system is mandatory for successful antibiotic therapy. PBMC simulation assays have shown potent immune activation on stimulation with C. Burnettii. Here we assess whether this holds true in situ by combining 3 six-marker panels with phenotypical markers describing the adaptive and innate immune system and looking at key check-points in immune regulation. Methods (Tissue) PCR-positive CQF infected AAAs (n=10) are compared to atherosclerotic AAAs (n=13) and normal abdominal aortas from kidney transplant donors (n=3). We combine DAPI and elastin autofluorescence with 3 distinct panels of 6 markers in an 8-color IHC technique (Vectra 3, Perkin Elmer, MA) to describe the local immune landscape. The panels focus on 1) adaptive immune response, 2) innate immune response and 3) checkpoint inhibitors. Cells were phenotyped and quantified using an automated quantitative analysis method. Results In contrast to normal abdominal aortas, both atherosclerotic AAAs and CQF AAAs show impressive lymphocyte recruitment and proliferation with the formulation of very large tertiary lymphoid structures. CQF AAAs show a 2-fold increase in regulatory T cell numbers compared to atherosclerotic AAAs. These regulatory cells are both surrounding germinal centers and are seen throughout the tissue. Additionally, CQF AAAs show more positive cells and a higher expression of PD-L1, which is a key immunosuppressive mediator. Conclusion The immune landscape within the aortic wall shows far greater immune suppression than previously found in in vitro and peripheral blood studies. We show that CQF infections are associated with severely increased numbers of regulatory T cells, even compared to the low-grade and chronic inflammation present in atherosclerosis. Subsequently, a look at the immune checkpoint PD-L1 shows an increased expression in CQF aortas, elucidating a possible underlying mechanism and treatment target. Figure 1. Representative microscopy images of normal (left) atherosclerotic AAA (middle) and CQF AAA (right), full thickness overview (top row) and magnified region off interest (bottom row). Vast increase in regulatory T cells (FoxP3+), recognisable by the yellow nucleus, surrounding germinal centres in only the CQF aorta. Furthermore, DAPI in blue, B cells in green, helper T cells in red, cytotoxic T cells in cyan, classic DC type 2 in magenta. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): ERC starting grant (ERC-2014-StG-336454-CoNQUeST); TTW-NWO open technology grant (STW-14716)