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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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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