Aspergillus fumigatus-Specific T Cells in Patients with Chronic Rhinosinusitis

Introduction: Aspergillus fumigatus belongs to the saprophytic fungi, and its spores form a significant part of the daily load of fungal spores inhaled as particles in aerosols. A. fumigatus is a possible T-cell activator. Its contribution to the pathogenesis of chronic rhinosinusitis (CRS) is controversially discussed. The aim of this study was to detect and characterize A. fumigatus-specific CD4+ and CD8+ T cells in patients with CRS with (CRSwNP) and without (CRSsNP) nasal polyps. Methods: Tissue and blood samples were collected from patients who underwent paranasal sinus surgery due to CRSwNP or CRSsNP. Afterward, purified CD4+ and CD8+ cells were cultured together with antigen-presenting cells. A peptide mix derived from A. fumigatus antigen was added to the cultures. After 6 days, multicolor flow cytometry was performed, and proliferation was measured using the marker Ki-67. Cytokine secretion was quantified from the supernatant of the cell culture. Results: Significant differences in the proliferation of nasal CD4+ T cells to A. fumigatus antigen were observed for cells from patients with CRSwNP in comparison to CRSsNP, while no differences were found between nasal and peripheral blood T cells. The activation of tissue-derived CD4+ T cells was associated with significantly higher concentrations of IL-4, IL-5, and IL-17a in the cell culture from patients with CRSwNP in comparison to CRSsNP and/or healthy controls. Conclusion: Our findings indicate that patients with CRSwNP harbor a higher proportion of A. fumigatus-reactive CD4+ T cells in the nasal mucosa than patients with CRSsNP. A. fumigatus-reactive CD4+ T cells of CRSwNP patients secreted TH2 cytokines and IL-17. Our findings suggest a role for A. fumigatus in the pathogenesis of CRSwNP and provide a rationale for targeted therapies.


Introduction
Chronic rhinosinusitis (CRS) is an inflammation of the nasal mucosa with two or more symptoms persisting for more than 12 weeks [1]. The main symptoms are nasal obstruction, anterior or posterior rhinorrhea, loss of sense of smell, facial pain, and cephalgie [1]. These symptoms have a high impact on the quality of life of CRS patients [2]. The disease can be divided into two subtypes according to phenotypical characteristics: CRS with (CRSwNP) or without nasal polyps (CRSsNP). A more distinct classification is based on the type of inflammation in the infected nasal tissue. Tomassen et al. [3] differentiate between several endotypes of CRS. In this study, tissue samples from patients with CRS were examined for type 1, type 2, and type 3 inflammation markers and then assigned to 10 different clusters. Here it could be shown that especially the patients with a severe nasal polyposis with frequent recurrences and a comorbid bronchial asthma have a much accentuated type 2 inflammation. About 80% of patients with nasal polyps in Europe show a predominant type 2 inflammation. However, in many cases, there is a mixture of different inflammatory markers, and a clear demarcation of inflammation types is not possible. This classification is more complex, and the development of certain endotypes is still not completely understood. Multiple external factors can influence the imbalance in the local tissue immune reaction. This cluster classification is still considered incomplete due to the pronounced heterogeneity of the CRS.
Fungi are omnipresent in our environment and commonly found in the nasal mucosa of patients with CRS. Aspergillus fumigatus belongs to the saprophytic fungi and its spores represent a significant part of the fungal spores, which we inhale every day as particles in aerosols [4]. Due to their size, A. fumigatus conidia avoid the mucociliary clearance of the respiratory tract, and it is postulated that adults inhale more than 100 A. fumigatus conidia daily [4].
It is well known that some fungi can act similarly to bacteria as sources of antigen for T-cell activation. Moreover, Staphylococcus aureus has been identified as a producer of superantigens in CRSwNP patients [5]. Among fungi, especially A. fumigatus is well described as a T-cell activator in different diseases [6]. The local nasal mucosa is highly colonized with a variation of fungi, and especially the presence of A. fumigatus was confirmed by current sequencing methods [7]. Currently, it has not yet been shown whether this colonization with A. fumigatus has an influence on the local inflammatory composition of patients with CRS, so there is a need to investigate this more closely. The aim of this study was, thus, to detect A. fumigatus-specific T cells in CRSwNP and CRSsNP patients and measure cytokine secretion of these cells after activation in vitro. The hypothesis of the preliminary study is an increased activation of the tissue T cells by A. fumigatus antigens as a possible trigger of the pathogenesis of CRS.

Ethical Issues
The Ethics Board of the Medical Faculty of the Julius-Maximilian-University Würzburg (No. 116/17) approved this study and all participants gave written informed consent.

Inclusion and Exclusion Criteria
The indication for paranasal sinus surgery was determined according to the European guidelines [1]. All patients received guideline-compliant conservative therapy, and in case of non-response, the indication for surgery was provided. The classification into the corresponding phenotypes (CRSwNP and CRSsNP) was made based on the endoscopic preoperative, intraoperative, and radiological findings. All patients were enrolled in the study between December 2017 and October 2018 at the Department of Otorhinolaryngology, Plastic, Aesthetic, and Reconstructive Head and Neck Surgery of the University of Würzburg (Würzburg, Germany). Patients with Churg-Strauss syndrome, primary ciliary dyskinesia or cystic fibrosis were excluded.

Histological Analyses
Specimens of 3 samples (one of each study group) were formalin fixed and embedded. 1 μm thick sections from formalin-fixed paraffin-embedded material were used for hematoxylin-eosin (H&E). Routine histological examination was performed.

Preparation of Human Lymphocytes
Human lymphocytes were obtained by venous puncture from patients undergoing paranasal sinus surgery and transferred to the laboratory as previously described [8,9]. Lymphocytes were separated by density-gradient centrifugation (10 min, 1,000 g) at room temperature (RT) on equal amounts of Ficoll (Biochrom, Berlin, Germany), using a membrane-containing 50-mL cell tube (Greiner Bio-One, Frickenhausen, Germany). Cell number and viability were determined by a Cell Counter + Analyzer System (CASY TT; Innovatis, Reutlingen, Germany) according to the manufacturer's protocol. After centrifugation with 500 g, cells were frozen at −80°C with 1 mL freezing medium containing 10 parts of fetal calf serum (LINARIS, Dossenheim, Germany) and one part of DMSO (Roth, Karlsruhe, Germany).

Preparation of Tissue Samples
All tissue samples were collected intraoperatively from patients undergoing regular paranasal sinus surgery due to CRSwNP (n = 10) or CRSsNP (n = 8). Additionally, healthy nasal mucosa from the inferior turbinate was collected from patients undergoing septoplasty with turbinoplasty due to non-inflammatory reasons (n = 7). The Multi Tissue Dissociation Kit and the gentleMACS Dissociator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were used for dissociation of the tissue samples according to the manufacturer's protocol. The obtained cell suspension was filtered through a mesh (sizes 100 to 40 μm) together with 15  with 500 g, cells were frozen at −80°C in 1 mL freezing medium containing 9 parts of fetal calf serum (LINARIS) and one part of DMSO (Roth, Karlsruhe, Germany).

Cell Sorting of CD4+, CD8+, and CD3− Antigen-Presenting Cells
Cell sorting was performed as positive selection of CD4+ and CD8+ T cells and negative selection of CD3− antigen-presenting cell (APC) by the MACS cell separation system with human CD3+, CD4+, and CD8+ Microbeads (Miltenyi Biotec GmbH) according to the manufacturer's protocol. A Cell Counter + Analyzer System (CASY TT; Innovatis) determined the amount of CD4+ and CD8+ T cells. CD3− APC were counted using the Neubauer cell chamber.
Cell Culture with A. fumigatus Peptide Mix 96-well round-bottom microplates were used for all cell culture experiments. 2.5 × 10 4 CD4+ or CD8+ T cells, 5 × 10 4 APC, 200 μL TexMacs Medium supplemented with 1% Penicillin/Streptomycin (Miltenyi Biotec GmbH) and 30 IU/mL IL-2 were added per well together with 1 μg/mL of a peptide mix from A. fumigatus (Peptivator A. fumigatus pmp20 research grade, Miltenyi Biotec GmbH). As a positive control, 2 μL of human T-activator CD3/CD28 Dynabeads (Miltenyi Biotec GmbH) was added instead of the A. fumigatus peptide mix. Cultures were incubated for 6 days at 37°C/5% CO 2 . After 6 days in culture, cells were pelleted by centrifugation (500 g, 5 min) and the Dynabeads were removed by magnetic separation before supernatants were collected.

FACS Analysis
Proliferation of CD4+ and CD8+ T cells was measured by the expression of the intracellular marker Ki-67 as previously described [10]. In short, the following antibodies were used: anti-CD45 Pacific Orange (Thermo Fisher Scientific Inc., Waltham, MA, USA), anti-CD3 phycoerythrin (PE)-Cy7, anti-CD4 Pacific Blue, anti-CD8a Alexa 700 (all BioLegend, Inc., San Diego, CA, USA), and anti-Ki-67 (BD Bioscience, San Jose, CA, USA). Mouse-IgG PE (BD Bioscience) served as an isotype control. Apoptotic cells were excluded through staining with Viability Dye 780 (eBioscience; Thermo Fisher Scientific Inc., Waltham, MA, USA). For intracellular staining of Ki-67, cells were treated with fixation buffer (eBioscience; Thermo Fisher Scientific Inc.). All antibodies were used according to the manufacturer's protocol. FACS analysis was performed using an LSR II flow cytometer (Becton, Dickinson and Company, San Diego, CA, USA), and the data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA). The gating strategy is shown in Figure 1.

Cytokine Secretion of Stimulated T Cells
The cytometric bead array Legendplex TM Human Cytokine Panel (13-plex) (Biolegend, San Diego, CA, USA) was used to measure cytokine secretion according to the manufacturer's protocol. Supernatants of cell cultures were collected after 6 days and frozen at −80°C until the multiplex assay was carried out. Thawed supernatants were mixed with capture beads, washing steps and addition of biotinylated detection antibodies and streptavidinphycoerythrin (SA-PE) followed. Measurement was performed using a FACS Canto (Becton, Dickinson and Company). Analysis of the cytokine concentrations of IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17a, IL-17f, IL-21, IL-22, IFN-γ, and TNF-α were performed with the Legendplex TM data analysis software (Biolegend).

Statistics
Data are presented as means ± standard deviation (SD) or as box plots depicting medians, 50% percentiles and range. Due to homogeneity of variance and the presence of a normal distribution, the statistical significance was analyzed by one-way ANOVA with Holm-Sidak test using GraphPad Prism Software 6.0c. Values of p < 0.05 were considered statistically significant.

Results
Patients' Characteristics 25 patients were included in the study. The mean age of all patients was 43.38 ± 18.61 years. Patients were enrolled in the study between December 2017 and October 2018 at the Department of Otorhinolaryngology, Plastic, Aesthetic, and Reconstructive Head and Neck Surgery of the University of Würzburg (Würzburg, Germany). Some of the patients included in the study already have presurgery on the paranasal sinuses due to their CRS. These  Table 1 under previous surgery. No significant differences between the variables of the study groups were measured. Patient characteristics are summarized in Table 1.

Histological Analyses
In 3 samples, immunhistological staining was additionally performed. In keeping with the pathophysiology of CRSwNP, a significant increase in eosinophilic granulation, goblet cell hyperplasia, and a thickened lamina propria can be seen here (Fig. 2a). The exemplary picture of the CRSsNP shows as an indication of a chronic and low-active inflammation transitional epithelium, in comparison, a significantly thinner basal membrane and no increase in eosinophilic granulocytes (Fig. 2b). The staining of the control group shows an inconspicuous picture with regular epithelium and without a significant increase in inflammatory cells (Fig. 2c).

TH2 Cytokine Secretion of A. fumigatus-Specific T Cells
For TH2 cytokines, significant differences were only measured for CD4+ T cells (Fig. 6b). No differences were found regarding CD8+ T cells (Fig. 6b). IL-4 had a significantly higher concentration in the culture supernatant of nasal CD4+ T cells from patients with CRSwNP (2.82 ± 2.0.2 pg/mL) in comparison to CRSsNP (0.95 ± 0.53 pg/mL, Between the control group and CRSsNP patients, no significant differences were observed. Similarly, IL-5 secretion by nasal CD4+ T cells of CRSwNP patients (302.53 ± 315.69 pg/mL) was significantly higher than that of CD4+ T cells of CRSsNP patients (9.20 ± 12.00 pg/mL, p = 0.036). No differences were found among the other groups.

Subgroup Analysis of A. fumigatus-Specific CD4+ T Cells from CRSsNP Patients
In contrast to nasal CD4+ T cells from CRSwNP patients, nasal CD4+ T cells of CRSsNP patients did not differ from CD4+ T cells from healthy controls (Fig. 5, 6). As we noted high interindividual differences regarding the T-cell responses to stimulation by A. fumigatus antigen, we subdivided the CRSsNP samples into responders and non-responders based on the proportion of Ki-67+ cells after stimulation with A. fumigatus antigen (Fig. 7a). A cutoff value of 5% Ki-67+ T cells was defined. Differences in the secretion of TH2 cytokines between responders and non-responders seemed to exist (Fig. 7b). However, evaluation of statistical differences was not possible due to the low number of samples in each group.

Discussion
The pathogenesis of CRS is still controversially discussed and considered multifactorial. The role of fungi and the influence of fungal colonization in chronic airway diseases remain unclear in this context. An early study by Ponikau et al. proposed that fungi are present in the nasal mucosa of mostly all patients with CRS [11]. Further studies showed no specific T-cell responses to fungi [12], and a major role in the pathogenesis [13] was revised. However, a negative effect on the inflammation of the nasal sinus mucosa by T-cell regulation is not conclusively clarified [12].
The aim of this study was to investigate whether the high local presence of A. fumigatus has a direct influence on the activation status of local T cells and thus on the immune response in CRS patients. In summary, significant differences in A. fumigatus-specific local CD4+ T cells of patients with CRSwNP in comparison to CRSsNP were observed. Differences between local and peripheral blood T cells were not found. The activation of tissue-derived CD4+ T cells from patients with CRSwNP in comparison to CRSsNP or healthy controls led to significantly higher concentrations of IL-4, IL-5, and IL-17a in cell culture supernatants. These findings indicate that in some patients with CRSwNP, tissue CD4+ T cells can be specifically activated by A. fumigatus antigen and stimulated to produce cytokines, especially TH2 cytokines.
Airway epithelium is continuously in direct contact with A. fumigatus. Innate immunity is responsible for elimination of spores and fungal material and for the induction of T-cell responses. TH2 T-cell memory responses to A. fumigatus are described to be pathologic and lead, in contrast to TH1/TH17 responses, to ongoing inflammation in the nasal mucosa. Chaudhary et al. [14] proposed that A. fumigatus antigens do not per se act as allergens because specific T-cell responses by TH1, TH2, and TH17 cells were also found in PBMC from healthy donors without any allergy. But, whether an anti-allergic TH1/TH17 or a pro-allergic TH2 response will occur is mostly likely dependent on a combination of intrinsic properties of the patients' T cells and the local inflammatory milieu within the tissue [14]. However, the inflammatory profiles of CRS are well described. It is known that in the majority of patients in Europe, a TH2 profile is predominant in CRSwNP and a Th1/Th17 profile in CRSsNP. Due to the heterogeneity of the diseases, also a mixed picture is possible [15,16], and a high variety among geographic regions has also been described [17]. In the present study, A. fumigatusspecific T cells were mainly isolated from CRSwNP patients and secreted IL-4 and IL-5.
Earlier, Fokkens et al. [13] rejected the hypothesis that fungi play an important role in the pathogenesis of CRS compared to other factors because there is no evidence of clinical improvement through antifungal drug therapy. The lack of a response toward antifungal therapy, further, suggests that during clinically overt CRSwNP, A. fumigatus-specific CD4+ T cells may not, or not primarily, be activated through detection of fungal antigen by the T-cell receptor. This, in turn, means that A. fumigatus-specific CD4+ T cells may undergo "bystander" activation during CRSwNP mediated by toll-like receptor agonists or cytokines [18]. Cytokinemediated activation of human memory CD4+ T cells has been shown to be sufficient to also induce release of the TH2 cytokine IL-4 [19].
Vogel et al. [20] evaluated age-related T-cell-specific responses to A. fumigatus, and strong cytokine answers to A. fumigatus antigens were already observed in neonates and infants. The responsive T-cell pool showed a significantly higher variability with 20 TCR-V β families in the neonatal [20]. This variability decreases significantly in adults. On the one hand, A. fumigatus can lead to lethal invasive infections and, on the other hand, for example, trigger allergic asthma in adults. TH1 CD4+ T cells are primarily responsible for the defense against invasive aspergillosis, while TH2 CD4+ T cells lead to an exacerbation of asthma [21]. Rivera et al. [22] were also able to show that live fungal spores are more likely to produce a Th1 response, while avital fungal spores produce a Th2 response. The question arises if the ubiquitous contamination of the nose with A. fumigatus in CRSwNP may induce T-cell memory in early life with a subsequent continuous TH2 inflammation in adult patients. This might be a reason why antifungal medication does not improve the symptoms of the disease. Consequently, it would be of particular interest if these specific CD4+ T cells could be reprogrammed in order to restore them to their original state or to switch them to a TH1/TH17 inflammatory pathway. Engineered T cells are a new promising target in antitumor therapy but also in several infectious diseases or in autoimmunity [23]. Even in CRSwNP, engineered T cells that can reduce the local inflammatory reaction and lead to a switch in the local T-cell memory subset might be a possible new therapeutic option. Due to the easy accessibility of the nasal mucosa, maybe a local and not a systemic therapy with reduced side effects is imaginable. Here, a form of specific immunotherapy (SIT) successfully used for decades to treat wasp venom and other allergies [24] could be implemented to treat CRSwNP. In order to be able to do so, the antigens recognized by the pathogenic T cells need to be identified. The results of this study thus pave the way toward SIT by identifying A. fumigatus-specific CD4+ T cells in the nasal mucosa of CRSwNP patients. We used a peptide pool derived from pmp20 (Asp f3) of A. fumigatus. For PBMC of healthy blood donors, it has been shown that addition of intact Asp f3 protein was processed and presented to T cells inducing secretion of IFN-γ, IL-4, and/or IL-17 [14]. Therefore, either intact Asp f3 or Asp f3-derived peptides might constitute suitable antigens for SIT in CRSwNP patients.
The presented study has several limitations. First, this pilot study only includes a low number of individuals. A higher number of subjects should be included in further studies to balance the high individual differences. Second, all patients with CRS received at least topical steroids prior to surgery in accordance with European guidelines [1]. Of course, this immunosuppressive therapy can be expected to have relevantly reduced cytokine secretion and reactivity of the T cells. Third, measurement of healthy controls must be analyzed critically because the total number of obtained local T cells from this non-inflamed tissue is lower in comparison to inflamed nasal mucosa and especially to nasal polyps. Therefore, the differences seen in this study on a per-cell basis between T cells of healthy controls and CRS patients might underestimate the true significance for CRS pathology. Fourth, a classification of the samples in this study according to the endotype classification of Tomassen et al. [3] was not carried out due to the small number of samples. However, it can be assumed that about 80% of patients with nasal polyps in Europe have a type 2 inflammation. However, in order to confirm the investigations in this pilot study, it is of great importance and should be part of further investigations to describe the precise A. fumigatus antigen-specific T-cell activation by depending on the present endotypes.
In summary, A. fumigatus-specific local CD4+ T cells were found in CRSwNP patients together with increased levels of IL-4, IL-5, and IL-17a secretion. These findings suggest an evident role of A. fumigatus-specific T cells in the pathogenesis of this disease and reiterate the search for targeted therapies for these patients.

Statement of Ethics
The Ethics Board of the Medical Faculty of the Julius-Maximilian-University Würzburg (No. 116/17) approved the study and all participants gave written informed consent.