Differences in local immune cell landscape between Q fever and atherosclerotic abdominal aortic aneurysms identified by multiplex immunohistochemistry

Abstract

Background: Chronic Q fever is a zoonosis caused by the bacterium Coxiella burnetii which can manifest as infection of an abdominal aortic aneurysm (AAA). Antibiotic therapy often fails, resulting in severe morbidity and high mortality. Whereas previous studies have focused on inflammatory processes in blood, the aim of this study was to investigate local inflammation in aortic tissue.Methods: Multiplex immunohistochemistry was used to investigate local inflammation in Q fever AAAs compared to atherosclerotic AAAs in aorta tissue specimen. Two six-plex panels were used to study both the innate and adaptive immune system.Results: Q fever AAAs and atherosclerotic AAAs contained similar numbers of CD68+ macrophages and CD3+ T cells. However, in Q fever AAAs the number of CD68+CD206+ M2 macrophages was increased, while expression of GM-CSF was decreased compared to atherosclerotic AAAs. Furthermore, Q fever AAAs showed an increase in both the number of CD8+ cytotoxic T cells and CD3+CD8-FoxP3+ regulatory T cells. Lastly, Q fever AAAs did not contain any well-defined granulomas.Conclusions: These findings demonstrate that despite the presence of pro-i is associated with an immune suppressed micro environment.Funding: This work was supported by SCAN consortium: European Research Area - CardioVascualar Diseases (ERA-CVD) grant [JTC2017-044] and TTW-NWO open technology grant [STW-14716].

Topography of immune cell infiltration in different stages of coronary atherosclerosis revealed by multiplex immunohistochemistry

“…LAD samples and tonsils as positive control were stained with two multiplex (mIHC) panels, defining adaptive and innate immune cells ( Table 1 ). Gorris et al described the optimization and validation of mIHC panels, and our mIHC method on vascular tissue has been described previously [21] , [22] . The principle behind our multiplex method is automatically staining of our samples with six consecutive tyramide signal amplification (TSA) stains followed by antigen retrieval and removing of the antibody complex in a Leica Bond system (BOND-Rx Fully Automated IHC and ISH, Leica Biosystems).…”

“…Regions for multispectral imaging on 20x magnification were selected (Phenochart 1.1; Akoya biosciences, MA). Images were unmixed using spectral libraries and inForm Advanced Image Analysis software (inForm 2.4.8; Akoya biosciences, MA), as described previously [22] . In summary, after training scanned slides were segmented in tissue, background, and blood, based on DAPI and autofluorescence, and in the innate panel, MMP9 and GMCSF were also used for nerve segmentation.…”

“…This processing resulted in 20x magnification images with tissue and single cell data (localization, tissue, phenotype, and marker) per slide, which were combined into single flow cytometry (fcs) files for analysis in FlowJo (FlowJo 10.0.7, Becton Dickinson, NJ). Quantification of cell types and subsets was performed as shown in Supplementary Figure 1 and as described previously [22] . Since we noticed that the expression of FoxP3 declined in post-mortem samples, this marker was not included in our analysis.…”