Q fever is a zoonosis caused by the Gram-negative intracellular bacterium Coxiella burnetii (C. burnetii), with natural reservoirs in a wide range of wild and domestic animals. In infected animals (such as goats) milk, placenta, and birth fluids, can contain this microorganism, which may cause human infections via inhalation. Acute Q fever can present as pneumonia, hepatitis, and isolated fever, yet 60% of cases are asymptomatic (Maurin and Raoult, 1999). Progression to chronic Q fever occurs in approximately 5% of infected individuals Fournier et al., 1998; people at risk are older, suffer from aortic or iliac aneurysm, or renal insufficiency (Kampschreur et al., 2012), or previously underwent valvular or vascular prosthesis surgery (Kampschreur et al., 2012). Chronic Q fever manifests as endocarditis or vascular Q fever, that is, infection of an abdominal aortic aneurysm (AAA) or vascular prosthesis (Wegdam-Blans et al., 2011; Botelho-Nevers et al., 2007; van Roeden et al., 2019).

Vascular manifestations of Q fever can have severe clinical consequences. In a population of proven and probable vascular manifestations of Q fever patients according to the Dutch consensus guideline, a Dutch cohort study has described that complications had occurred in 61% of the cases. Of these, acute complications (i.e., rupture, dissection, endoleak, or symptomatic aneurysms) were most prevalent (35%), followed by abscesses (22%), and fistula (14%). Moreover, 25% of patients had a definitely or probably chronic Q fever related cause of death (van Roeden et al., 2019). In addition, serological screening of 770 patients with aorto-iliac disease, for example, aneurysms or previous vascular reconstructions, demonstrated that 16.9% was seropositive for Q fever, of which 30.8% suffered from chronic Q fever. In this group, aneurysm-related acute complications were more common than in aneurysm patients without Q fever (Hagenaars et al., 2014b).

To elucidate the pathology underlying chronic Q fever, previous studies have mainly focussed on immune responses in peripheral blood. Blood mononuclear cells of patients with chronic Q fever, when exposed to C. burnetii in vitro, produce high amounts of Interferon-gamma (IFNg), the proinflammatory cytokine considered crucial for killing of the pathogen (Schoffelen et al., 2014; Schoffelen et al., 2017). In the infected tissues, C. burnetii resides and replicates in monocytes and macrophages (Ghigo et al., 2009). In vascular manifestations of Q fever, it is assumed that C. burnetii survives in resident macrophages in the vascular wall (Lepidi et al., 2009; Lepidi et al., 2003). In such patients, there is an apparent inability to effectively eradicate C. burnetii, despite the aforementioned IFNg response. In general, a pro-inflammatory response with granuloma formation and intracellular killing or control of the bacterium by activated ‘M1’ monocytes/macrophages is required to contain intracellular infections like Q fever. Surprisingly, the apparent inability of chronic Q fever patients to kill C. burnetii has only been investigated in studies in vitro which showed suggestive roles for polarization to tolerogenic M2 macrophages and increased numbers of circulating regulatory T cells (Layez et al., 2012; Benoit et al., 2008).

It is still unclear how C. burnetii survives and locally escapes the immune system in vascular manifestations of Q fever. To address this, we have investigated the local immune response in C. burnetii-infected AAAs (Q fever AAA), classical atherosclerotic AAA, acutely infected AAA, and control aorta tissue, applying multiplex immunohistochemistry (mIHC) on human patient tissues. We investigated both the adaptive and innate immune systems. We show that in vascular manifestations of Q fever numerous immune suppressive mechanisms appear to be present, including the absence of pro-inflammatory granulomas, increased numbers of regulatory T cells, polarization of macrophages into the tolerogenic M2 phenotype, and decreased expression of GM-CSF.

Abdominal aorta tissue samples from patients with Q fever infected aneurysms and control groups (i.e., atherosclerotic AAAs, acutely infected AAAs, and non-aneurysmatic aortas) were investigated with a novel mIHC method to study the involvement of the innate and adaptive immune system in vascular manifestations of Q fever. The data underlying this article will be shared upon reasonable request to the corresponding author.

Imaging, multispectral unmixing, and analysis

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After staining, image acquisition and immune cell quantification were performed using an automated approach. First, the PerkinElmer Vectra (Vectra 3.0.3; PerkinElmer, MA) scanned whole slides at 4× magnification and 20× magnification, allowing precise cell segmentation incorporating the entire sample. The average 20× views per slide was 283 resulting in an average tissue area of 59.0±32.6 mm², and this high number substantially reduces the chance of sampling bias. Spectral libraries and inForm Advanced Image Analysis software (inForm 2.4.8; Akoya Biosciences, MA) unmixed these multispectral images (Figures 1A and 2A).

Figure 1

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Figure 2

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Subsequently, inForm Advance Imaging Analysis software was used for segmentation of tissue and cells. For tissue segmentation, tissue slides were divided into tissue, infiltrate, thrombus, blood, and background (Figure 1C). This segmentation was based on DAPI, autofluorescence and, if present, also CD20 and CD3. For single-cell segmentation, cells were identified with DAPI and autofluorescence, and, depending on the panel, with membrane markers CD20 and CD3 (Figure 1B) or CD68, CD206, and CD15. Requiring DAPI for cell segmentation ensured the exclusion of artifact staining, in which DAPI is absent. The output of the software was 20× magnification images and cell data (localization, tissue, phenotype, and marker) per slide. Images were combined into single flow cytometry standard (fcs) files, allowing analysis in FlowJo (FlowJo 10.0.7, Becton Dickinson, NJ). In FlowJo, only cells in tissue and infiltrate were analyzed and gates were drawn as shown in Figure 1E for the adaptive panel and Figure 2B for the innate panel by two observers with excellent interobserver correlation (Figures 1D and 2D).

These clear distinct positive cell populations were not found for CD45RO and MMP9 as their expression is gradual (Booth et al., 2010). For that matter, the gates for these markers were drawn in negative populations, namely non-T cells and non-neutrophils, respectively. Following this, these gates were copied to populations that could express these markers. GM-CSF fcs files did not show distinct positive and negative populations although they were visible in the microscopy images. Therefore, inForm Advance Imaging Analysis software was used for automatic thresholding for GM-CSF per sample, providing the number of GM-CSF positive pixels per sample (Figure 2C).

We are the first to introduce mIHC in vascular manifestations of Q fever to study ongoing local inflammation. This sophisticated mIHC method enabled us to quantify immune cells in large sections of tissue which minimized sampling bias. First, we showed that granulomas are absent in Q fever AAAs. Second, atherosclerotic and Q fever AAAs were similar when comparing the numbers of immune cells. However, there were striking differences in the composition of macrophage- and T cell-phenotypes between AAAs and Q fever AAAs, leading to new insights into the pathogenesis of vascular manifestations of Q fever and its complications and possibly with therapeutic consequences.

Our first observation, the absence of well-formed granuloma formation in our cohort of Q fever AAAs is an important one, since it suggests that the local immune landscape lacks an adequate pro-inflammatory response. In our series, we could not find any well-formed granuloma similar to how they are described in acute Q fever. In acute Q fever manifesting in non-vascular tissue, so-called doughnut granulomas are reported: granulomas with a central clear space and a fibrin ring within or at its periphery (Maurin and Raoult, 1999; Faugaret et al., 2014), for example, in liver biopsies in case of hepatitis (Pellegrin et al., 1980). Here, granuloma is a feature of active defense against the pathogen. However, we should take into account that our group of acute aortitis with Streptococcus species did not show granulomas either. In chronic Q fever granulomas have not been described before (Faugaret et al., 2014; Raoult et al., 2005). In particular, Lepidi described that resected valve specimens of patients with Q fever endocarditis lacked well-formed granulomas (Lepidi et al., 2003). In vascular Q fever, granulomatous responses consisting of histiocytes surrounding necrotic areas have been reported in Q fever AAAs, however, well-formed granulomas were not found (Hagenaars et al., 2014a). Thus, we would interpret the absence of organized granulomas as the first clue for an immune-suppressed environment in AAA of Q fever patients that allows persisting infection after the acute phase.

Second, our results demonstrate some similarities between Q fever AAAs and AAAs. Percentages of CD3⁺ T cells, CD20⁺ B cells, CD1c⁺ cDC2, CD15⁺ neutrophils, and CD68⁺ macrophages are similar between the groups with atherosclerotic AAA and Q fever AAA. This finding is supported by the PCA, which shows overlapping populations of atherosclerotic and Q fever AAAs when entering these inflammatory cell markers. This does not come as a surprise since there are suggestions that vascular manifestations of Q fever develop in preexisting atherosclerotic aneurysms (Botelho-Nevers et al., 2007; Hagenaars et al., 2014b; Hagenaars et al., 2014a; Broos et al., 2015; Eldin et al., 2017).

Despite the similarities, we discovered that atherosclerotic and Q fever AAAs do have important differences, which emerge when investigating macrophage and T cell subset markers. Macrophages in Q fever AAAs were found to be polarized into the less inflammatory M2 phenotype, which is ‘tolerogenic’ and poorly microbicidal, in contrast to the M1 phenotype that possesses a machinery that can clear an infection. Interestingly, in the AAAs, we found less M2 polarization based on the presence of CD206. This would either indicate that macrophages polarize toward M2 in response to C. burnetii infection, or that the presence of M2 polarization is a prerequisite for C. burnetii persistence. Previous studies have demonstrated that C. burnetii inhabits and proliferates in monocytes and macrophages, and more specifically, in resident vascular wall macrophages in case of vascular Q fever (Ghigo et al., 2009; Lepidi et al., 2003). It has been shown by Benoit et al., 2008 that C. burnetii stimulates an atypical M2 activation program in monocyte-derived macrophages in vitro (Benoit et al., 2008). M2 polarization of macrophages was also observed in C. burnetii infected transgenic mice constitutively expressing IL-10 in macrophage lineage, a mouse model for chronic Q fever pathogenesis (Meghari et al., 2008). Spleens and livers of these mice showed increased expression of arginase-1 and mannose receptor (CD206) and decreased expression of iNOS, IL-12, and IL-23 in bone marrow-derived macrophages after infection with C. burnettii compared to C. burnetii-infected wild-type mice. These previous findings suggested that chronic Q fever is associated with M2 polarization of macrophages, but direct evidence in chronic Q fever patients was lacking. Our findings establish that outgrowth and persistence of C. burnetii in AAAs is associated with the predominance of CD68⁺CD206⁺ M2 macrophages.

There are several possible explanations for the lack of macrophage activation. First, our results demonstrate decreased expression of GM-CSF in Q fever AAAs compared to AAAs. GM-CSF is a pro-inflammatory cytokine that activates granulocytes and macrophages (Hercus et al., 2012). Its decreased expression in Q fever AAAs may contribute to the immune-suppressive environment in Q fever AAA. The role of GM-CSF in the context of aneurysm formation has been investigated previously (Son et al., 2015). Strikingly, Son et al. described the increased occurrence of aortic dissection and intramural hematoma in wild-type mice subjected to aortic inflammation (CaCl2+ Ang II administration) when also receiving GM-CSF. Only administrating GM-CSF, without the prerequisite of aortic inflammation, did not result in aortic dissection or intramural hematoma. Its potential clinical relevance was confirmed in human blood: GM-CSF serum levels of patients suffering from acute dissection were higher than controls with coronary artery disease, aortic aneurysms or healthy volunteers (Son et al., 2015). Additionally, in our cohort, we found that Q fever AAAs ruptured at smaller diameter compared to atherosclerotic AAA. This finding, combined with the GM-CSF paradox, suggests that the development of Q fever AAAs and atherosclerotic AAAs follow different pathways, however strictly hypothetically.

Second, a key cytokine in activation is IFNg, a T-helper (Th)-1 cytokine that activates macrophages and makes them more microbicidal. Previous studies from our group have demonstrated that peripheral blood mononuclear cells from patients with chronic Q fever exhibit an abundant production of IFNg when exposed to C. burnetii antigens (Schoffelen et al., 2014; Schoffelen et al., 2017). These findings were enigmatic since there is an apparent inability of the patient’s immune system to kill C. burnetii at the infected sites. The current findings would be compatible with a downregulated IFNg response at the infected site.

In addition to differences in macrophage subsets, differences in T cell subsets were also observed. Numerous T cells were observed both in tissue and in TLS, of which the latter are known ectopic lymphoid tissues at inflammation sites, including infections and auto-immune diseases such as atherosclerosis (Akhavanpoor et al., 2014; Akhavanpoor et al., 2018; Jing and Choi, 2016). These structures are important inductive sites for T cells and antibody production (Jing and Choi, 2016; Polverino et al., 2016; Humby et al., 2009). The first difference is the number of CD3⁺CD8⁺ cytotoxic T cells, which increased in both infiltrate and surrounding tissue of Q fever AAA compared to AAA. Although the numbers of cytotoxic T cells were high, their function might be compromised, resulting in defective elimination of C. burnetii. The increased numbers of CD3⁺CD8⁻FoxP3⁺ regulatory T cells we found in Q fever AAAs may play a role here. An increased number of circulating regulatory T cells has also been shown by Layez et al., 2012 in Q fever endocarditis patients and in acute Q fever patients (Layez et al., 2012). Regulatory T cells can inhibit cytotoxic T cells directly or indirectly (Joosten and Ottenhoff, 2008), with a possible role for IL-10 produced by this T cell subset. An important role of IL-10 in chronic development of Q fever has been postulated based on converging evidence from a series of in vitro studies. IL-10 production by peripheral blood mononuclear cells from patients with Q fever endocarditis and Q fever with valvulopathy who were at risk for developing chronic Q fever was high, compared to control individuals (Capo et al., 1996; Honstettre et al., 2003). Moreover, IL-10 specifically increases C. burnetii replication in naive monocytes (Ghigo et al., 2001) possibly by downregulating IFNg. Finally, low IL-10 production in monocytes from patients with acute Q fever was associated with C. burnetii elimination, whereas C. burnetii replicated in monocytes from patients with chronic Q fever and high IL-10 production. The microbicidal activity of monocytes from patients with chronic Q fever was restored by neutralizing IL-10 (Ghigo et al., 2004). The murine model of chronic Q fever mentioned above, also confirmed a key role for IL-10 in bacterial persistence. C. burnetii infection is persistent in mice that overexpress IL-10 in the macrophage compartment (Meghari et al., 2008). Thus, IL-10 could play a crucial role in this immune-suppressed environment.

The last major difference between Q fever AAA and atherosclerotic AAA is the extent of damage to the vascular wall architecture in Q fever AAAs. This is demonstrated by extensive loss of elastin fibers and increase of fibrosis present in the vascular wall. Fragmentation of elastin fibers has been described for AAAs previously (Coady et al., 1999); however, we found the loss of elastin fibers more evident in the lesions from Q fever AAAs than atherosclerotic AAAs. Fibrosis is characterized by replacement of normal tissue by excessive connective tissue and usually follows chronic inflammation. Presence of fibrosis is a sign of a type 2 immune response (Gieseck et al., 2018), which we also demonstrated in our cohort with the abundance of M2 macrophages. This may be the effect of persistent presence of growth factors, proteolytic enzymes, angiogenic factors, and profibrotic cytokines (Wynn, 2008; Wynn, 2007). Previously, fibrosis was also observed in chronic Q fever endocarditis in humans and cows (Lepidi et al., 2003; Agerholm et al., 2017; Brouqui et al., 1994; De Biase et al., 2018). This indicates that our Q fever AAA cohort suffered from more destructive disease than our AAA cohort.

These novel insights could lead to new clues for novel treatments and thus developments for clinical care. Currently, Q fever AAA still leads to significant morbidity and mortality rates despite antibiotic and surgical treatment. Epidemiological studies demonstrate the similar risk profile of Q fever and non-Q fever infected AAAs, yet the risk of complications is higher in the Q fever infected group (Hagenaars et al., 2014b), even up to 61% (van Roeden et al., 2019), and 25% of patients suffering from Q fever AAA had deceased with a definitely/probably chronic Q fever related cause of death (van Roeden et al., 2019). Here, we confirm that in vascular manifestations of Q fever, the local immune response is skewed toward an immunotolerant state. Hypothetically, the decreased expression of GM-CSF suggests a possible role for immunomodulating treatment, for example, with administration of recombinant GM-CSF. This is already approved for neutropenia due to myelosuppression (Dougan et al., 2019), and has been suggested for treatment for pulmonary tuberculosis (Damiani et al., 2020). There might be a role for immunomodulating adjuvant therapies in patients with Q fever AAA in whom treatment failure is observed with antibiotics alone.

Our study was the first to use mIHC in Q fever AAA and thereby to gain information about the number and proportion of immune cells, and simultaneously obtain spatial information. This powerful technique and the access to rare Q fever AAA tissue are strengths of this study. While other studies have tested for immune cell activation and recruitment in peripheral blood, we were able to study the actual infected tissue. Interpreting our results in context of previous observations enables us to increase our understanding of the pathophysiology of Q fever AAA. Still, several limitations should be noted. First, our sample size is limited with only 10 vascular manifestations of Q fever samples. However, this is still the largest study investigating local immune responses in Q fever AAA in humans. In addition, in our quantification method, we include entire slides up to 238 20× views per patient, which minimizes the effects of the small sample size. Second, consistent with IHC studies in general, we can only describe the immune cells we observe, without answering mechanistic questions. Although epidemiological studies suggest that Q fever AAA is the result of C. burnetii infected AAAs, our study did not support causation. Moreover, our study lacks direct IHC identification of C. burnetii in Q fever AAA, however, all samples were proven PCR positive. Additionally, elastin (breakdown) and fibrosis were not quantified. Finally, it should be emphasized that these results and interpretations are based on tissue samples from patients with indication for surgery. Nevertheless, when interpreting our results in light of the current literature, we can reasonably formulate hypotheses about the pathophysiology and test these in further research.

Taken together, this leads to the following hypothesis with a prominent role for immune suppression. First, macrophages that harbor C. burnetii are not effectively killing the micro-organisms, probably due to a lack of activation by proinflammatory cytokines like GM-CSF and IFN-g in a microenvironment with excess IL-10. Second, effector T cells that attempt to eliminate the intracellular bacterium residing in monocytes and macrophages, are hindered by regulatory T cells that are prominent IL-10 producers. Third, there is a lack of microbicidal M1 macrophages, instead macrophages are polarized into the tolerogenic M2 phenotype, which leads to insufficient attack of the pathogen, enabling persistent infection.

All data generated or analyzed during this study are included in the manuscript and uploaded to Dryad (http://dx.doi.org/10.5061/dryad.bzkh189b4). Figure 3 - Source data 3; Figure 5 - Source data 5; Figure 6 - Source figure 6; Figure 7 - Source figure 7 contain numerical data used to generate the figures.

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Decision letter after peer review:

Thank you for submitting your article "Multiplex immunohistochemistry differences between Q fever and atherosclerotic abdominal aortic aneurysms indicate immune suppression" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Roland Lang (Reviewer #1); Jean-Louis Mege (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Although the authors worked with two 6-marker panels, the extent of multiplexing in the IHC methodology is still limited. Thus, the combination of antibodies used to define immune cell types and subsets or activation states has a limited resolution and leaves out populations of interest. E.g. the differentiation between M2 and M1 macrophages is exclusively made based on CD206 staining; no staining for CD4 T cells (not to speak of Th subsets) was included. Similarly, only GM-CSF was the only cytokine measured. The authors should be careful about defining these populations on the limited markers, for example, macrophages polarization can not only be based on differential expression of CD206. Can other markers be added/used? Otherwise, conclusions and interpretations need to be tempered accordingly.

2) A blind spot in the authors' visual field of the Q fever aneurysm is spatial and quantitative information about the pathogen. While all samples were classified by PCR for C. burnetii as Q fever or control atherosclerotic aneurysms, no IHC of C. burnetii is included in the mIHC panels or in a separate analysis. Thus, information about the types and activation status of C. burnetii-harboring host cells is absent: are the M2-like CD206+ macrophages infected more heavily? Is there a spatial association of immunoregulatory cells (Foxp3+ Treg) with infected host cells? Are C. burnetii also infecting endothelial cells or neutrophils? Is it possible to add staining for C. burnetii in the aneurysms?

3) An inherent limitation of the descriptive nature of the study is that it cannot determine a causal role for specific findings in the development of vascular Q fever. E.g., the higher abundance of M2-like macrophages may be a cause or a consequence of chronic C. burnetii infection. The finding of reduced GM-CSF and increased CD8⁺ T cells in Q fever aneurysms is novel and interesting, but the functional relevance thereof remains unclear. Therefore, it is better to avoid causal conclusions like the last sentence of the abstract.

4) The lack of quantification for elastin destruction and fibrosis (Figure 6) is surprising since it does not keep up with spirit of the rest of the manuscript. The authors should provide also in this Figure a quantitative assessment of fibrosis and elastin breakdown. Development of fibrosis is a hallmark of type 2 immune bias (e.g. liver fibrosis in schistosoma infection). Given the abundance of M2-like macrophages in the aortic tissue in vascular Q fever, I wonder whether this potential link to elastin breakdown and fibrosis should not be made in the Discussion section.

5) The authors did not observe granuloma structures in the Q fever aneurysms. This finding is consistent with earlier reports on the histopathology of heart valves in chronic Q fever. It is stressed in the manuscript that the lack of granulomas is indicative of the immunosuppressive environment. However, the comparison with the hepatic, dough-nut granulomas in acute infection is problematic because of the completely different tissue types. The correct comparison would in fact be to use aortic tissue from another infection and to determine whether granulomas are formed in the vessel wall. Was there formation of granulomas in the aneurysms from acute bacterial infection?

6) Is it clear that all Q fever aneurysms are the result of super-infection of existing atherosclerotic aneurysms with C. burnetii, as presented in the Discussion? Would this statement not require the demonstration of the aneurysms prior to C. burnetii infection? How strong is the evidence to exclude that aneurysm formation is a consequence of C. burnetii infection?

7) The title provides a partially correct conclusion of the results. If there is a signature in favor of M2 polarization in Q fever AAA, this is not the demonstration of immune suppression. The authors should re-write the title to better represent the findings of the manuscript. The authors should also temper similar claims throughout the manuscript.

8) The authors should describe how they calculated the number of patients necessary to conduct the study. In addition, the proportion of men in infectious AAA is only 50% whereas it was 83% in atherosclerotic AAA. Is this a consequence of the small size of infectious AAA sample. What is specific to infectious AAA as compared with atherosclerosis AAA?

9) The authors should quantify tertiary lymphoid structures and describe why they consider them as a measure of lymphocyte proliferation.

The small size of infectious AAA sample is a source of conclusion. It would be better to include these results in additional material.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Differences in local immune cell landscape between Q fever and atherosclerotic abdominal aortic aneurysms identified by multiplex immunohistochemistry" for further consideration by eLife. Your revised article has been evaluated by a Reviewing Editor and a Senior Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1. Line 64: " suffer from renal insufficiency or aneurysm," This sentence is factually incorrect since the kidney does not develop aneurysms – only blood vessels do. The authors need to indicate what they mean (likely aortic aneurysm) and also provide a corresponding reference.

2. Line 69: The sentence "Vascular Q fever has severe clinical consequences" is not quite accurate, since not all patients are affected equally. It should be rephrased to "Vascular manifestations of Q fever can have severe clinical consequences". Also, since "vascular Q fever" also includes heart valves (which was not studied here) it should also be replaced with "vascular manifestations of Q fever" in lines 66, 83, 93, 97, 104, 264, 302, 307, 331, 342, and 430.

3. Line 71: "acute aneurysms". There is no such thing as an "acute aneurysm". However, patients with aortic aneurysms may experience acute complications, such as rupture which carries a high mortality. This needs rewording.

4. Line 93 ff: The authors do not indicate that the aneurysm tissue studied actually were obtained from human patients, which greatly increases the clinical importance and implications of this work.

5. Line 101 ff: There is no information about the "control" group patients, how the tissues were obtained, whether they had aortic atherosclerosis, aneurysms or not etc. A description of gross pathology of the specimens needs to be included for each patient studied, e.g. in a table. Also, for all groups the age of the patients included needs to be provided. This information is essential and needs to be added.

6. Line 289 "Q fever aortas reveal extensive fibrosis" This is a remarkable and novel finding which at this point cannot be explained by the locally suppressed micro-environment observed by the author, and actually would be counterintuitive. The authors should discuss this rather paradoxical finding with regard to the potential mechanisms involved.

Essential revisions:

1) Although the authors worked with two 6-marker panels, the extent of multiplexing in the IHC methodology is still limited. Thus, the combination of antibodies used to define immune cell types and subsets or activation states has a limited resolution and leaves out populations of interest. E.g. the differentiation between M2 and M1 macrophages is exclusively made based on CD206 staining; no staining for CD4 T cells (not to speak of Th subsets) was included. Similarly, only GM-CSF was the only cytokine measured. The authors should be careful about defining these populations on the limited markers, for example, macrophages polarization can not only be based on differential expression of CD206. Can other markers be added/used? Otherwise, conclusions and interpretations need to be tempered accordingly.

We totally agree with the reviewers regarding cell (subset) populations. When we designed these panels, we decided to create panels for studying the overview of immune cells. More in-depth characterization of macrophages and T helper cells and their cytokines would be an excellent follow-up study.

We are aware of the limitations of the chosen markers in this manuscript. We acknowledge that we cannot distinguish separate helper T cell populations and that CD206 is not the only marker for macrophage polarization. Therefore, we made substantial revisions to our manuscript in the results and Discussion sections.

Page/line number: page 7 line 17; page 8 line 5-24; page 11 line 12-13; page 12 line 2; page 13 line 69.

2) A blind spot in the authors' visual field of the Q fever aneurysm is spatial and quantitative information about the pathogen. While all samples were classified by PCR for C. burnetii as Q fever or control atherosclerotic aneurysms, no IHC of C. burnetii is included in the mIHC panels or in a separate analysis. Thus, information about the types and activation status of C. burnetii-harboring host cells is absent: are the M2-like CD206+ macrophages infected more heavily? Is there a spatial association of immunoregulatory cells (Foxp3+ Treg) with infected host cells? Are C. burnetii also infecting endothelial cells or neutrophils? Is it possible to add staining for C. burnetii in the aneurysms?

We thank the reviewer for this valuable comment. We agree that spatial information about the pathogen would be a valuable addition to this manuscript. We would like to emphasize that the samples are PCR positive, which is a very sensitive method for proving the presence of C. burnetii. Literature applying IHC for C. burnetii is scarce, and after discussion with experts, also from other centers, we concluded specific antibodies were available (1), but unfortunately, not anymore. In literature some stainings have been used, amongst others the Kinyoun and Auramine method (2, 3). Both are methods to demonstrate acid-fast bacteria. Kinyoun is a modified Ziehl-Neelsen staining and visible in brightfield, Auramine-Rhodamine is a fluorescent staining visible in the Texas Red channel.

In optimization of these experiments, we noticed that using the Kinyoun method, identification of the bacteria was very difficult because of their small size and poor contrast to the surrounding tissue. Therefore we decided to stain our tissue using the Auramine method, since fluorescence provides better contrast, which would help us in finding the bacteria in the Kinyoun-stained tissue. During optimization of the Auramine experiments, we noticed that the IHC and DAPI were influenced by the auramine staining, and order experiments have pointed out that the best results were gained by (1) IHC followed by (2) DAPI and scanning, and then the same sample underwent (3) Auramine staining and repeatedly (4) DAPI followed by the final scan. Additionally we only applied Auramine staining on a consecutive slide.

Author response image 1 shows the positive control, which is a lymph node from a goat with an acid-fast bacterium inhabiting. It is clear that Auramine is in high numbers present in both methods of staining.Author response image 2 shows a magnification of an intima layer of the aorta which shows only one Auramine-positive spot. This spot is not colocalizing with CD68. Additionally, only treating the sample with Auramine-staining on a consecutive slide did not reveal more positive areas. When we use this information to study the Kinyoun staining, we did not find any bacterium-like structures. Author response image 3, shows a magnification of an adventitia layer of the aorta. Here are some more Auramine-positive spots but again, these are scarce compared to our positive control. In the Kinyoun staining we could not find bacterium-like structures.

Author response image 1

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Author response image 2

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Author response image 3

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As a negative control, we included one or our normal aortas. Unfortunately, in this tissue we also found a Auramine-positive structure (Author response image 4), with the same shape as shown before.

Author response image 4

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Concluding: we attempted to identify C. burnetii implementing staining techniques as described in literature but we were not able to obtain reliable results, as also mentioned in literature. We believe that PCR is the best option at this point of time and we mention the lack of direct staining in the limitations. We think that these results might give directions for future research but currently we are very cautious drawing conclusions. Additionally, it is known that IHC of pathogens involved in chronic infections can be very challenging, as demonstrated earlier in tuberculosis. We have added the limitation of lacking of C. burnetii direct staining in the limitations. Page/line number: page 15 line 13-14.

3) An inherent limitation of the descriptive nature of the study is that it cannot determine a causal role for specific findings in the development of vascular Q fever. E.g., the higher abundance of M2-like macrophages may be a cause or a consequence of chronic C. burnetii infection. The finding of reduced GM-CSF and increased CD8⁺ T cells in Q fever aneurysms is novel and interesting, but the functional relevance thereof remains unclear. Therefore, it is better to avoid causal conclusions like the last sentence of the abstract.

We thank the reviewer for this comment. We acknowledge that we cannot draw causal conclusions about disease progression and development. Therefore, we have made changes in our abstract and discussion to avoid these causal statements. Page/line number: page 3 line 15-16; page 16 line 12-14.

4) The lack of quantification for elastin destruction and fibrosis (Figure 6) is surprising since it does not keep up with spirit of the rest of the manuscript. The authors should provide also in this Figure a quantitative assessment of fibrosis and elastin breakdown. Development of fibrosis is a hallmark of type 2 immune bias (e.g. liver fibrosis in schistosoma infection). Given the abundance of M2-like macrophages in the aortic tissue in vascular Q fever, I wonder whether this potential link to elastin breakdown and fibrosis should not be made in the Discussion section.

We want to thank the reviewer for this comment. We agree that quantification is the best method of presenting our data. However there were difficulties in quantification of fibrosis, which hampered us from this analysis. We have presented an overview of this issue in Author response image 5 in this response letter below. Even at high level magnification and use of both HE and EVG staining, it was not possible to accurately draw borders between fibrosis and non-fibrotic tissue. In addition, we considered additional stainings to address this question. However, fibrotic tissue and healthy adventitia both consist of collagen and thus a fibrosis staining, showing the presence of collagen, is not able to distinguish between pre-existent and fibrotic adventitia and will not solve the problem of the difficulty of drawing borders between fibrotic and non-fibrotic tissue. Nevertheless, although we were not able to quantify this, this finding was too interesting not to describe in this manuscript and is still supportive to the main message, and hopefully this could inspire future work.

Author response image 5

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We would like thank the reviewer for the excellent comment regarding the type 2 immune bias in relation to M2 macrophages. We have added this valuable argument to the discussion. Page/line number: page 14 line 6-8.

Secondly, we also would like to address the second part of the reviewer’s comment, which is the quantification of elastin fibers and/or destruction. We completely agree that quantification of this described trend would be a valuable addition for our manuscript. Therefore we attempted to quantify the amount of elastin fibers in our samples based on the EVG stained images. To do so, we have tried different methods as shown in Author response image 6 and Author response image 7.

Author response image 6

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Author response image 7

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Author response image 6 demonstrates the analysis in inForm, which is the software in which we have performed all other analyses. inForm allows so-called tissue segmentation, in which regions are drawn for training, and after training the algorithm will segment all images based on this specific training. Image 4ABC demonstrate some representative training regions and the segmentation after training on these specific images. In these examples the different regions (background, tissue, and elastin) are recognized well. However, Author response image 6 is more representative for the majority of the images. Not all elastin fibers are recognized well which results in underestimation of the presence of these fibers.

Therefore we decided to continue our analysis in FIJI. Our protocol is based on literature describing brightfield threshold analysis. (4) First, the image of interest Author response image 7A was converted into RGB format Author response image 7. To be able to perform threshold analysis, color deconvolution needed to take place for which this plugin was downloaded (https://blog.bham.ac.uk/intellimic/g-landinisoftware/colour-deconvolution-2/). However, EVG staining was not an option in the color deconvolution menu, thus we have selected several ‘second best’ options Author response image 7. These deconvoluted images contained the highest contrast between elastin and non-elastin. After applying the Threshold Tool on all of these images, we noted that in all samples there was no optimal threshold which included (almost) all elastin without excluding (almost) all cells in the infiltrate.

In conclusion: both the inForm and FIJI method did not provide the reliable elastin quantification we were aiming for. Therefore, we decided not to include any of these analyses in our manuscript but we still believe that this trend is an interesting addition to our manuscript. We made changes to the limitations section to acknowledge the lack of the quantification of fibrosis and elastin. Page/line number: page 15 line 14-15

5) The authors did not observe granuloma structures in the Q fever aneurysms. This finding is consistent with earlier reports on the histopathology of heart valves in chronic Q fever. It is stressed in the manuscript that the lack of granulomas is indicative of the immunosuppressive environment. However, the comparison with the hepatic, dough-nut granulomas in acute infection is problematic because of the completely different tissue types. The correct comparison would in fact be to use aortic tissue from another infection and to determine whether granulomas are formed in the vessel wall. Was there formation of granulomas in the aneurysms from acute bacterial infection?

Thank you for your comment. Indeed, the comparison with acutely infected aortic tissue would be better than the non-related tissue. We have implemented this suggestion in the Discussion section. Page/line number: page 10 line 13-17.

6) Is it clear that all Q fever aneurysms are the result of super-infection of existing atherosclerotic aneurysms with C. burnetii, as presented in the Discussion? Would this statement not require the demonstration of the aneurysms prior to C. burnetii infection? How strong is the evidence to exclude that aneurysm formation is a consequence of C. burnetii infection?

The reviewer remarks an excellent point. We do think that epidemiological studies can support this statement, however, we agree that there not conclusive evidence supporting this statement. We made adjustments to the Discussion section accordingly.

Page/line number: page 12 line 5, page 15 line 12-16.

7) The title provides a partially correct conclusion of the results. If there is a signature in favor of M2 polarization in Q fever AAA, this is not the demonstration of immune suppression. The authors should re-write the title to better represent the findings of the manuscript. The authors should also temper similar claims throughout the manuscript.

We would like to thank the reviewer for this comment. Throughout the manuscript and in the title, we have tempered claims about immune suppression. Page/line number: page 1 line 1-2, page 3 line 15-16, page 11 line 12-13, page 12 line 2, page 15 line 12-16.

8) The authors should describe how they calculated the number of patients necessary to conduct the study. In addition, the proportion of men in infectious AAA is only 50% whereas it was 83% in atherosclerotic AAA. Is this a consequence of the small size of infectious AAA sample. What is specific to infectious AAA as compared with atherosclerosis AAA?

We would like to thank the reviewer to point out this important issue. We agree that proper sample size calculation should be performed in order to execute robust statistical analysis.

In our study, we included rare tissue samples. Therefore we included all available samples for these rare groups: normal abdominal aortas, Q fever AAA and acute infection of AAA.

To determine how many atherosclerotic AAAS should be included, we performed a sample size calculation based on preliminary data. This calculation is added to the ‘Transparent Reporting Form’.

Moreover, the difference between infectious AAA and atherosclerotic AAA is the PCR proven presence of pathogen in the infectious AAA.

We are aware that there may be a bias in the included samples: only subjects are included with an indication for surgery, this applies to all four groups. We have added the sample size calculation to the and a description of this potential bias to the limitations in the Discussion section. Page/line number: page 15 line 15-16.

9) The authors should quantify tertiary lymphoid structures and describe why they consider them as a measure of lymphocyte proliferation.

The small size of infectious AAA sample is a source of conclusion. It would be better to include these results in additional material.

We would like to thank the reviewer for this comment. We agree that TLS should be quantified, so we have added this information to the Results section. In addition we have added additional information about TLS in the Discussion section and the definitions for TLS in the method section. We agree that the sample size of infectious AAA is very limited. We clarified immune cell infiltration in infectious AAA in figure 1, so we believe that further clarification in the supplemental section is not needed anymore.

References

1. Roest H-J, van Gelderen B, Dinkla A, Frangoulidis D, van Zijderveld F, Rebel J, et al. Q fever in pregnant goats: pathogenesis and excretion of Coxiella burnetii. PloS one. 2012;7(11):e48949-e.

2. Leyk W, Scheffler R, Schliesser T. [Direct demonstration of Coxiella burnetii from air samples on slides coated with kraton 1107]. Zentralbl Bakteriol Orig A. 1976;234(1):105-9.

3. Moore JD, Barr BC, Daft BM, O'Connor MT. Pathology and diagnosis of Coxiella burnetii infection in a goat herd. Vet Pathol. 1991;28(1):81-4.

4. Surman TL, Abrahams JM, Manavis J, Finnie J, O’Rourke D, Reynolds KJ, et al. Histological regional analysis of the aortic root and thoracic ascending aorta: a complete analysis of aneurysms from root to arch. Journal of Cardiothoracic Surgery. 2021;16(1):255.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1. Line 64: " suffer from renal insufficiency or aneurysm," This sentence is factually incorrect since the kidney does not develop aneurysms – only blood vessels do. The authors need to indicate what they mean (likely aortic aneurysm) and also provide a corresponding reference.

We like to thank the reviewer for this sharp observation. We have changed the sentence accordingly and we have added a reference as follows:

“Progression to chronic Q fever occurs in approximately 5% of infected individuals (2); people at risk are older, suffer from aortic or iliac aneurysm or renal insufficiency (3), or previously underwent valvular or vascular prosthesis surgery. (3)”

2. Line 69: The sentence "Vascular Q fever has severe clinical consequences" is not quite accurate, since not all patients are affected equally. It should be rephrased to "Vascular manifestations of Q fever can have severe clinical consequences". Also, since "vascular Q fever" also includes heart valves (which was not studied here) it should also be replaced with "vascular manifestations of Q fever" in lines 66, 83, 93, 97, 104, 264, 302, 307, 331, 342, and 430.

Thank you for this comment, we agree that adjustment of this sentence and term improves the manuscript. We changed this sentence into ‘Vascular manifestations of Q fever can have severe clinical consequences’ and replaced ‘vascular Q fever’ by ‘vascular manifestations of Q fever’ throughout the manuscript. Line number 66, 67, 81, 91, 96, 103, 263, 264, 301, 307, 330, 417, 430.

3. Line 71: "acute aneurysms". There is no such thing as an "acute aneurysm". However, patients with aortic aneurysms may experience acute complications, such as rupture which carries a high mortality. This needs rewording.

We would like to thank the reviewer for this comment. We have reworded this sentence into:

“Of these, acute complications (i.e. rupture, dissection, endoleak or symptomatic aneurysms) were most prevalent (35%), followed by abscesses (22%), and fistula (14%).” Line number 69.

4. Line 93 ff: The authors do not indicate that the aneurysm tissue studied actually were obtained from human patients, which greatly increases the clinical importance and implications of this work.

Thank you for this comment, this is indeed an important addition which we have added to the study description as follows:

“To address this, we have investigated the local immune response in C. burnetii-infected AAAs (Q fever AAA), classical atherosclerotic abdominal aortic aneurysms (AAA), acutely infected AAA, and control aorta tissue, applying multiplex immunohistochemistry (mIHC) on human patient tissues. We investigated both the adaptive and innate immune system.” Line number 94-95.

5. Line 101 ff: There is no information about the "control" group patients, how the tissues were obtained, whether they had aortic atherosclerosis, aneurysms or not etc. A description of gross pathology of the specimens needs to be included for each patient studied, e.g. in a table. Also, for all groups the age of the patients included needs to be provided. This information is essential and needs to be added.

Thank you for pointing out this important issue. To further clarify these groups, we have added a small introduction in line 102, which now introduces the patient groups as following:

“Abdominal aorta tissue samples from patients with Q fever infected aneurysms and control groups (i.e., atherosclerotic AAAs, acutely infected AAAs, and non-aneurysmatic aortas) were investigated with a novel mIHC method to study the involvement of the innate and adaptive immune system in vascular manifestations of Q fever.”

The paragraph ‘Patient Samples’ (line 108-122) describes the groups in more detail and we have added some information about the obtainment of these samples. The first three groups (Q fever AAA, atherosclerotic AAA, and acutely infected AAA) are all aneurysmatic aortic tissue samples, contrary to the normal non-aneurysmatic aortas. Our atherosclerotic AAAs were selected at random from our database. Tissue of acutely infected AAAs and non-aneurysmatic aortas is rather scarce and because of their limited availability, we have included all available and eligible samples for our study.

This information has now been added to this paragraph, resulting in the following:

“Patient samples

Tissue samples were collected from four groups of patients in two Dutch hospitals: Jeroen Bosch Hospitals in ‘s Hertogenbosch and Radboud university medical center in Nijmegen. The first group consisted of patients diagnosed with C. burnetii infected AAA (Q fever AAA) according to the Dutch consensus guideline (15): all patients had an abdominal aneurysm (AAA) and IgG phase I was at least 1:1024 in combination with a positive PCR of aortic tissue. The second group consisted of patients with atherosclerotic AAA without clinical suspicion of Q fever which were selected from out database at random. The third group consisted of patients with an acutely infected AAA, with the same definition of AAA in combination with positive cultures of Streptococcus pneumoniae and Streptococcus Agalactiae, respectively. In these three groups AAA was defined as a CT proven abdominal aortic aneurysm with a diameter of at least 3.0 cm. (16) All aneurysmatic tissue samples were either obtained from patients undergoing elective surgical repair or emergency repair in case of aortic rupture. The fourth group consisted of abdominal aorta samples from patients undergoing kidney explantation surgery for transplantation purposes, with an aortic diameter smaller than 3.0 cm. Due to the limited availability of acutely infected AAAs and non-atherosclerotic aortas, all available and eligible samples were selected. The samples from Jeroen Bosch Hospital were described in a previous study. (17)”.

In addition, Table 1 contains descriptive information about all included groups (page 36). Here, we elaborate on sex, age, diameter on CT and various clinical baseline characteristics of all groups. As requested, we have added all ages to this table.

We hope that this information answers your question. Please let us know if we did not interpret your question correctly. By all means, we are willing to further improve our manuscript.

6. Line 289 "Q fever aortas reveal extensive fibrosis" This is a remarkable and novel finding which at this point cannot be explained by the locally suppressed micro-environment observed by the author, and actually would be counterintuitive. The authors should discuss this rather paradoxical finding with regard to the potential mechanisms involved.

We would like to thank the reviewer for this valuable comment. This extensive fibrosis is indeed an interesting finding which was discussed in the first revision. In the first revision the reviewers commented the following:

Development of fibrosis is a hallmark of type 2 immune bias (e.g. liver fibrosis in Schistosoma infection). Given the abundance of M2-like macrophages in the aortic tissue in vascular Q fever, I wonder whether this potential link to elastin breakdown and fibrosis should not be made in the Discussion section.

Based on this comment, we have further improved the paragraph elaborating on fibrosis in the Discussion section to the following:

“The last major difference between Q fever AAA and atherosclerotic AAA is the extent of damage to the vascular wall architecture in Q fever AAAs. This is demonstrated by extensive loss of elastin fibers and increase of fibrosis present in the vascular wall. Fragmentation of elastin fibers has been described for AAAs previously (23), however we found the loss of elastin fibers more evident in the lesions from Q fever AAAs than atherosclerotic AAAs. Fibrosis is characterized by replacement of normal tissue by excessive connective tissue, and usually follows chronic inflammation. Presence of fibrosis is a sign of a type 2 immune response (42), which we also demonstrated in our cohort with the abundance of M2 macrophages. This may be the effect of persistent presence of growth factors, proteolytic enzymes, angiogenic factors, and profibrotic cytokines. (43, 44) Previously, fibrosis was also observed in chronic Q fever endocarditis in humans and cows. (12, 45-47) This indicates that our Q fever AAA cohort suffered from more destructive disease than our AAA cohort.”

In this paragraph we elucidate the presence of fibrosis in context of potential mechanisms and literature describing fibrosis in chronic Q fever endocarditis in both human and bovine tissue. We believe that this section contains all potential perspectives on fibrosis development in Q fever AAAs. Please inform us if we misinterpreted this comment, so we can improve our manuscript accordingly.

Author details

1. Kimberley RG Cortenbach

   Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands

   Contribution

   Conceptualization, Formal analysis, Visualization, Writing – original draft

   Contributed equally with

   Alexander HJ Staal

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2717-5527

2. Alexander HJ Staal

   Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands

   Contribution

   Conceptualization, Formal analysis, Investigation, Writing – review and editing

   Contributed equally with

   Kimberley RG Cortenbach

   Competing interests

   No competing interests declared

3. Teske Schoffelen

   Department of Internal Medicine, Division of Infectious Diseases and Radboud Center for Infectious Diseases, Radboud University Medical Centre, Nijmegen, Netherlands

   Contribution

   Conceptualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. Mark AJ Gorris

   Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

5. Lieke L Van der Woude

   Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

6. Anne FM Jansen

   Department of Internal Medicine, Division of Infectious Diseases and Radboud Center for Infectious Diseases, Radboud University Medical Centre, Nijmegen, Netherlands

   Contribution

   Conceptualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

7. Paul Poyck

   Department of Surgery, Radboud University Medical Centre, Nijmegen, Netherlands

   Contribution

   Conceptualization, Resources, Writing – review and editing

   Competing interests

   No competing interests declared

8. Robert Jan Van Suylen

   Department of Pathology, Jeroen Bosch Ziekenhuis, 's Hertogenbosch, Netherlands

   Contribution

   Conceptualization, Resources, Writing – review and editing

   Competing interests

   No competing interests declared

9. Peter C Wever

   Department of Medical Microbiology and Infection Control, Jeroen Bosch Ziekenhuis, 's Hertogenbosch, Netherlands

   Contribution

   Conceptualization, Investigation, Resources

   Competing interests

   No competing interests declared

10. Chantal P Bleeker-Rovers

    Department of Internal Medicine, Division of Infectious Diseases and Radboud Center for Infectious Diseases, Radboud University Medical Centre, Nijmegen, Netherlands

    Contribution

    Conceptualization, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

11. Mangala Srinivas

    1. Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands
    2. Department of Cell Biology and Immunology, Wageningen University, Wageningen, Netherlands

    Contribution

    Funding acquisition, Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

12. Konnie M Hebeda

    Department of Pathology, Radboud University Medical Centre, Nijmegen, Netherlands

    Contribution

    Investigation, Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4181-3302

13. Marcel van Deuren

    Department of Internal Medicine, Division of Infectious Diseases and Radboud Center for Infectious Diseases, Radboud University Medical Centre, Nijmegen, Netherlands

    Contribution

    Conceptualization, Writing – review and editing

    Competing interests

    No competing interests declared

14. Jos W Van der Meer

    Department of Internal Medicine, Division of Infectious Diseases and Radboud Center for Infectious Diseases, Radboud University Medical Centre, Nijmegen, Netherlands

    Contribution

    Conceptualization, Supervision, Writing – review and editing

    Competing interests

    Senior editor, eLife

15. Jolanda M De Vries

    Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands

    Contribution

    Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

16. Roland RJ Van Kimmenade

    Department of Cardiology, Radboud University Medical Centre, Nijmegen, Netherlands

    Contribution

    Conceptualization, Methodology, Supervision, Writing – review and editing

    For correspondence

    Roland.vanKimmenade@radboudumc.nl

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8207-8906

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