Abstract Over 100 million women use progesterone therapies worldwide. Despite having immunomodulatory and repair properties, their effects on the outcome of viral diseases outside of the reproductive tract have not been evaluated. Administration of exogenous progesterone (at concentrations that mimic the luteal phase) to progesterone-depleted adult female mice conferred protection from both lethal and sublethal influenza A virus (IAV) infection. Progesterone treatment altered the inflammatory environment of the lungs, but had no effects on viral load. Progesterone treatment promoted faster recovery by increasing TGF-β, IL-6, IL-22, numbers of regulatory Th17 cells expressing CD39, and cellular proliferation, reducing protein leakage into the airway, improving pulmonary function, and upregulating the epidermal growth factor amphiregulin (AREG) in the lungs. Administration of rAREG to progesterone-depleted females promoted pulmonary repair and improved the outcome of IAV infection. Progesterone-treatment of AREG-deficient females could not restore protection, indicating that progesterone-mediated induction of AREG caused repair in the lungs and accelerated recovery from IAV infection. Repair and production of AREG by damaged respiratory epithelial cell cultures in vitro was increased by progesterone. Our results illustrate that progesterone is a critical host factor mediating production of AREG by epithelial cells and pulmonary tissue repair following infection, which has important implications for women’s health.

Author Summary Worldwide, the use of hormonal contraceptives is on the rise as a primary intervention for improving women’s health outcomes through reduced maternal mortality and increased childhood survival. There are many hormone contraceptive formulations, all of which contain some form of progesterone. Although the effects of hormone contraceptives and progesterone, specifically, have been evaluated in the context of infections of the reproductive tract, the effects of progesterone at other mucosal sites, including the respiratory tract have not been systematically evaluated. We have made the novel observation that administration of progesterone to female mice depleted of progesterone confers protection against both lethal and sublethal influenza A virus infection. In particular, progesterone reduces pulmonary inflammation, improves lung function, repairs the damaged lung epithelium, and promotes faster recovery following influenza A virus infection. Progesterone causes protection against severe outcome from influenza by inducing production of the epidermal growth factor, amphiregulin, by respiratory epithelial cells. This study provides insight into a novel mechanistic role of progesterone in the lungs and illustrates that sex hormone exposure, including through the use of hormonal contraceptives, has significant health effects beyond the reproductive tract.

Citation: Hall OJ, Limjunyawong N, Vermillion MS, Robinson DP, Wohlgemuth N, Pekosz A, et al. (2016) Progesterone-Based Therapy Protects Against Influenza by Promoting Lung Repair and Recovery in Females. PLoS Pathog 12(9): e1005840. https://doi.org/10.1371/journal.ppat.1005840 Editor: Stacey Schultz-Cherry, St. Jude Children’s Research Hospital, UNITED STATES Received: June 7, 2016; Accepted: August 2, 2016; Published: September 15, 2016 Copyright: © 2016 Hall et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by grants from the National Institutes of Health, National Institute of Allergy and Infectious Diseases (grant numbers AI112838 and HHSN272201400007C to SLK and AI097417 to AP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Hormonal contraceptives are listed as an essential medication by the World Health Organization (WHO)[1] because of the profound benefits these compounds can have on women’s health outcomes, including decreased rates of maternal mortality and improved perinatal outcomes and child survival, by widening the intervals between pregnancies [2]. Hormonal contraceptive formulations vary, but all contain some form of progesterone (P4) either alone or in combination with estrogen. There are currently over 100 million young adult women on P4-based contraceptives worldwide [3], with the WHO projecting that over 800 million women will be using contraceptives, including P4-based contraceptives, by 2030 [2]. Despite the staggering numbers of women taking these compounds, very few studies evaluate the impact of contraceptives on responses to infection or vaccination, especially in non-sexually transmitted diseases. Natural P4, produced by the ovaries during reproductive cycles, or synthetic P4 analogues found in contraceptives, signal primarily through progesterone receptors present on many cells in the body, including immune cells (e.g., NK cells, macrophages, dendritic cells (DCs), and T cells) as well as non-immune cells, such as epithelial cells, endothelial cells, and neuronal cells [4, 5]. Human, animal, and in vitro studies show that P4 can alter the immune environment and promote homeostasis by decreasing inflammation and inducing anti-inflammatory responses. For example, in the presence of P4, macrophages and DCs have a lower state of activation, produce higher levels of anti-inflammatory cytokines, such as IL-10, and produce lower amounts of proinflammatory cytokines, such as IL-1β and TNF-α, as compared with placebo treated cells [6, 7]. When either mice or cord blood cells from humans are treated with P4, the percentages of Foxp3+ regulatory T cells (Tregs) increase [8, 9]. Although the immunomodulatory effects of P4-based therapies in the form of contraception have been studied in the context of sexually transmitted infections, including HIV and herpes simplex virus [10–12], the impact of P4 on the outcome of viral infectious diseases outside of the reproductive tract has not been considered in either humans or animal models. Influenza A viruses (IAVs) primarily infect respiratory epithelial cells and induce the production of proinflammatory cytokines and chemokines that recruit immune cells, causing a local proinflammatory environment [13]. Infiltration and activation of CD4+ and CD8+ T cells, while necessary for the clearance of IAVs [13–15], can trigger inflammation and lead to tissue damage and severe outcomes from IAV infection [16]. Protection requires a balance between inflammatory responses generated to control virus replication and eliminate virus-infected cells with responses that mediate the repair of damaged areas of the lung. Repair involves a complex interplay among many cell types, cytokines, chemokines, growth factors, and extracellular matrix proteins that remodel tissue after acute injury, such as IAV infection [17]. Amphiregulin (AREG) is an epidermal growth factor that has emerged as a significant mediator of tissue repair at mucosal sites, including the lungs [18, 19], gastrointestinal tract [20, 21], and reproductive tract [22, 23]. Many immune cells produce AREG, but epithelial cells are the principle producer of AREG following inflammation or tissue injury [24]. If P4 can downregulate inflammatory immune responses and promote regulatory or tissue repair responses, then this hormone, at concentrations that reflect the luteal phase of the reproductive cycle, may improve the outcome of IAV infection. Epidemiological and experimental evidence suggest that young adult females suffer a worse outcome than males following IAV infection, which in mice is associated with infection-induced suppression of reproductive hormones and excessive inflammatory immune responses in females [25–27]. In addition to influenza, young adult females suffer a worse outcome than males from several autoimmune diseases, including multiple sclerosis [28]. Paradoxically, a growing body of literature reveals that exogenous treatment of females (both humans and mice) with either estrogens or P4 limits inflammation and protects against infectious and autoimmune diseases by decreasing inflammation and promoting repair [25, 29–31]. In this series of studies, we show that treatment with sustained physiological doses of P4 protects females against IAV by reducing inflammation and improving pulmonary function, primarily through upregulation of AREG in epithelial cells. The observation that P4 regulates the cellular and molecular mediators of tissue repair at a mucosal site outside of the reproductive tract to restore tissue homeostasis after infection or injury has broad implications for women’s health.

Discussion Hosts have evolved several mechanisms for overcoming viral infections, such as the induction of antiviral defenses that increase resistance to infection, or the activation of regulatory and repair responses that increase tolerance to the negative consequences of infection. In the present study, P4 significantly protected females during IAV infection by altering inflammation, improving pulmonary function, and promoting a pulmonary repair environment, which resulted in an earlier recovery. The protective effects of P4 were primarily mediated by the induction of AREG during both lethal and sublethal infections. Progesterone did not increase resistance to infection in females as demonstrated by the lack of an effect of P4 treatment on virus titers, clearance of infectious virus, numbers of Th1 cells, and CD8+ T cell activity in lungs. Instead, P4 reduced the detrimental consequences of IAV infection in females by increasing their tolerance to infection. Several host immunological factors, including TGF-β, Tregs, and regulatory populations of CD39+ Th17 cells, are associated with maintaining the balance between protective and pathological immune responses during IAV infection. Although P4 treatment had no effect on the numbers of Tregs in the lungs during IAV infection, concentrations of TGF-β and IL-6, the expression of Il23 and Il22, the number of Th17 cells, as well as the proportion of Th17 cells expressing CD39, were increased. Regulatory Th17 cells express the ectonucleotidase CD39 and are associated with repair following inflammation and infection [40, 41]. Th17 cells also promote epithelial cell proliferation and repair in the gut, primarily through the induction of IL-22 [38]. Consequently, treatment of females with P4 increased IL-22, a cytokine that has been shown to mediate regeneration of lung epithelial cells following IAV infection [43]. Whether the P4-induced increase in regulatory Th17 cells and IL-22 promotes cellular proliferation and repair of the lung epithelium during IAV infection by increasing AREG production requires consideration. Because P4 directly induced AREG production in respiratory epithelial cells in vitro, P4-induced AREG production may occur independent of the reparative effects of regulatory Th17 cells in the lungs during IAV infection. Progesterone induces repair of epithelial cells in the endometrium and myelin fibers in the central nervous system [44, 45]. This repair of myelin fibers by P4 [46] is one factor mediating how this reproductive hormone mitigates the progression of multiple sclerosis [29]. Our data show that P4 promotes proliferation of pulmonary cells, including epithelial cells, and pulmonary tissue repair. The reparative effects of P4 in the reproductive tract are caused by the induction of AREG, which promotes epithelial remodeling in mammary and uterine tissues [22, 23]. In the respiratory tract, AREG is involved in pulmonary tissue remodeling and repair during lung injury, asthma, and infection [18, 19, 21, 47, 48]. Although Areg-gene deficient mice show few abnormalities under homeostatic conditions [42], their ability to resolve inflammation or infection is severely impaired [20, 21]. During IAV infection, administration of rAREG protects mice from severe IAV-mediated morbidity by decreasing hypothermia, improving pulmonary function, and decreasing protein leakage into the airways [18, 19]. The data presented are the first report of P4 induction of AREG outside of the reproductive tract and in the context of infection. The effect of other reproductive hormones on AREG expression, including differential expression between males and females, warrants further study. AREG is produced primarily by epithelial cells [49], but type 2 innate lymphoid cells (ILC2) and Tregs have also been shown to produce AREG during IAV infection and contribute to the repair during resolution of infection [18, 19, 49, 50]. Because each of these cell type express progesterone receptors [5, 51], each is a potential producer of AREG in response to P4 treatment. Our in vivo and in vitro data suggest that respiratory epithelial cells are a predominant source of P4-induced AREG. Following IAV infection, AREG expression was predominantly localized to the bronchiolar epithelial cells, and P4 treatment of isolated mTECs increased AREG production following mechanical damage. Furthermore, P4-treatment did not activate markers of ILC2s, including IL-13 and IL-33 production, or increase numbers of Tregs in the lungs during infection, suggesting that the induction of AREG in response to P4 may not be occurring in these immune cell populations. Recovery following IAV infection is generally defined as a return of body temperature or body mass back to homeostatic levels [52]. In this study, however, we showed that pulmonary pathology and impaired pulmonary function persisted after measures of overall health, including hypothermia and clinical disease, returned to baseline. Furthermore, the impact of IAV infection was observed long after infectious virus had been cleared from the lungs. Recovery following IAV infection extended beyond 21 dpi and should be defined not only by reduced morbidity, but also by restored pulmonary function, both of which were expedited by P4 treatment in females. Progesterone concentrations fluctuate naturally during the female life span, with moderate concentrations during the menstrual cycle, high concentrations during pregnancy, and low concentrations following menopause. Progesterone is also used exogenously by over 100 million women worldwide in P4-based hormonal contraceptives, by post-menopausal women taking hormonal replacement therapy, and by both men and women in the treatment of cancer, osteoporosis, and brain injury [3, 53]. Prior to this study, the health consequences of P4-based therapies in acute respiratory infection had not been characterized. We have demonstrated that AREG, which is a significant factor that induces tissue repair and recovery from infectious diseases, is regulated by P4 during both lethal and sublethal IAV infection. The data presented provide critical mechanistic information about how P4 and possibly synthetic P4 analogues affect women’s health outside of the reproductive tract. Contraceptives that contain P4 are listed as an essential medication by the WHO, being a safe and effective method for improving health outcomes in women, including those living with HIV [1]. During outbreaks of infectious diseases that harm pregnant women and their fetuses (e.g., the current Zika outbreak), the WHO recommends increased use of hormonal contraceptives, which according to our data could have additional beneficial consequences on the outcome of other infectious diseases.

Materials and Methods Ethics statement All experiments were performed in compliance with the standards outlined in the National Research Council’s Guide to the Care and Use of Laboratory Animals. The animal protocol (M015H236) was reviewed and approved by the Johns Hopkins University Animal Care and Use Committee. All efforts were made to minimize animal suffering. Animals Adult (7–8 weeks old) female C57BL/6 mice were purchased from NCI Frederick. Areg+/- (C57BL/6 129 Sv) mice were kindly provided by Dr. Marco Conti (University of California San Francisco) and bred to obtain Areg-/- and Areg+/+ female littermates. Mice were housed 5 per microisolator cages under standard BSL-2 housing condition with food and water ad libitum. Surgical procedures At 8–12 weeks of age, mice were anesthetized with an intramuscular injection of ketamine (80 mg/kg) and xylazine (8 mg/kg) cocktail and ovaries were removed bilaterally as previously described [25]. All animals were given two weeks to recover prior to infection. Recombinant amphiregulin (10μg; R&D) was administered intraperitoneally every other day using saline as the vehicle. Hormone replacement and quantification Ovariectomized (ovx) mice were assigned to receive subcutaneous implants of placebo (-P4) or 15 mg progesterone (+P4) 21-day release pellets (Innovative Research of America) prior to IAV inoculation. Circulating concentrations of P4 were assessed from plasma using ether extraction and radiolabelled immunoassay, with P4 antibody (MP Biomedicals) and tracer 3H-P4 (American Radiolabeled). Uterine horns were removed at several time-points upon euthanasia of mice and wet weight was quantified as a bioassay for P4. Virus infection and quantification Mouse-adapted influenza A viruses, A/Puerto Rico/8/34 (PR8; H1N1) provided by Dr. Maryna Eichelberger at the Food and Drug Administration (FDA) and A/California/04/09 (ma2009; H1N1) generated by Dr. Andrew Pekosz from a published sequence [54], were used in these studies. Mice were anesthetized and inoculated intranasally with 30 μl of DMEM (mock) or H1N1 virus (1.78 50% mouse lethal dose (MLD 50 ) for PR8 and 0.4 MLD 50 for ma2009). Clinical disease scores for IAV-infected mice were based on four parameters, with one point given for each of the following: dyspnea, piloerection, hunched posture and absence of an escape response. For virus quantification, log 10 dilutions of lung homogenates (starting at 10−1) were plated onto a monolayer of MDCK cells in replicates of 6 for 4–6 days. Cells were stained with naphthol blue black (Sigma Aldrich) and scored for cytopathic effects. The 50% tissue culture infectious dose (TCID 50 ) was calculated according to the Reed-Muench method. Cytokine and chemokine quantification Snap-frozen lung tissue was homogenized in DMEM supplemented with 1% penicillin/streptomycin and 1% L-glutamine (Invitrogen) and centrifuged to remove cellular debris. Supernatants were harvested to measure IL-1β, TGF-β, IL-4, IL-5, IL-13, IL-17, IL-33, and AREG by ELISA (R&D Systems and BD Biosciences) and CCL-2, IL-12(p70), TNF-α, IFN-γ, IL-6 and IL-10 with the mouse inflammation cytometric bead array (BD Biosciences) according to the manufacturer’s protocols. Real time reverse transcription PCR Snap-frozen lung tissue or mTECs were homogenized in TRIzol and RNA was purified by chloroform extraction. RNA concentration and purity was measured using a NanoDrop (ThermoFisher Scientific). The RNA concentration in each sample was standardized to 1 μg using RNAse-free water. Reverse transcription was carried out using the iScript cDNA synthesis kit (Biorad) according to the manufacturer’s protocol. Pre-designed Il23 (Mm.PT.58.10594618.g), Il22 (NM_016971.2), Areg (Mm.PT58.31037760), Gapdh (Mm.PT.39a.1) and Pr (Mm.PT.58.10254276) PrimeTime Primers were purchased from Integrated DNA Technologies. Semi-quantitative RT-PCR was performed in 96-well optical reaction plates using the SsoFast EvaGreen Supermix (Biorad) on the StepOnePlus RT-PCR system (Applied Biosystems). Gene expression was normalized to Gapdh and mock-infected samples or wells with no injury using the ΔΔCt method. Flow cytometry analyses of T cells Lungs were excised and single-cell suspensions were generated following red blood cell lysis. Total viable cells were determined using a hemocytometer and trypan blue (Invitrogen) exclusion and resuspended at 1x106 cells/ml in RPMI 1640 (Cellgro) supplemented with 10% FBS (Fisher Scientific) and 1% penicillin/streptomycin. For IAV-specific T cells enumeration, cells were cultured for 5h with IAV peptide antigen (CD8:NP 366-374 , or CD4: HA 211-255 , NP 311-325, respectively) (ProImmune) in media containing Brefeldin A (GolgiPlug, BD) The viability of cells was determined by fixable Live/Dead violet viability dye (Invitrogen) and Fc receptors were blocked using anti-CD16/32A. The T cell populations were stained with the following antibodies: PerCP-Cy5.5 conjugated anti-CD4 (RM4-5)A, PerCP-Cy5.5 conjugated anti-CD8 (53–6.7)A, FITC conjugated anti-CD25 (7D4)A, PE conjugated DbNP 366-374 tetramer (NIH Tetramer Core Facility), FITC conjugated anti-CD4 (RM4-5)B, APC conjugated anti-CD3 (17A2B, and PerCP-eFluor 710 conjugated anti-CD39 (24DMS1)B. Intracellular staining with PE conjugated anti-TNF-α(MP6-XT22)A, FITC conjugated anti-IFN-γ (XMG1.2)A, PE conjugated anti-IL-4 (11B11)A, and PE conjugated anti-IL-17 (TC11-1810)A, was performed following permeabilization and fixation with Cytofix/Cytoperm and Perm/Wash bufferA. Intracellular staining with PE-conjugated Foxp3 (MF23)A was performed following fixation and permeabilization with a Foxp3 staining buffer setA. Data were acquired using a FACS Calibur (Cellquest Software) and analyzed using FlowJo (Tree Star, Inc.). Total cell counts were determined by multiplying each live cell population percentage by the total live cell counts acquired prior to staining by trypan blue exclusion counts on a hemocytometer. All reagents were purchased from BD BiosciencesA or eBioscienceB unless stated otherwise. Histopathology and immunohistochemistry Lungs were inflated, fixed in Z-fix (Anatech), embedded in paraffin, cut into 5μm sections, and mounted on glass slides. Slides were stained with hematoxylin and eosin (H&E) and used to evaluate lung inflammation. Histopathological scoring was performed by a single blinded veterinary pathologist on a scale from 0–3 (0, no inflammation; 1, mild inflammation; 2, moderate inflammation; and 3, severe inflammation) for the following parameters: bronchiolitis, alveolitis, vasculitis, perivasculitis, necrosis, consolidation, and edema [55, 56]. The sum of these parameters represents the cumulative inflammation score. The percentage of lesioned areas within each tissue section was also evaluated. Histopathological slides were deparafinized with xylene and rehydrated in graded ethanol. Heat-induced antigen retrieval with citrate buffer was performed and slides were blocked with 10% normal serum prior to overnight primary antibody incubation. For Ki67, rabbit anti-Ki67 (1/200; Abcam) was used, detected with the EXPOSE rabbit specific HRP/DAB detection kit (Abcam), counterstained with Hematoxylin and slides were mounted using Permount (Fisher). For immunofluorescence, anti-AREG (1/100; R&D) and anti-β-tubulin IV (1/100; BioGenex) were used and detected with appropriate secondary antibodies (1/400) conjugated to AF-555 (Thermo) and AF488 (Molecular probes). Slides were then treated against autofluorescence using 0.3% Sudan Black B (Sigma) in 70% ethanol and mounted using anti-fade medium containing DAPI (ProLong Gold from Cell Signaling Techonology). Images were taken using a Nikon Eclipse E800 (for H&E and Ki67 stains) or a Zeiss AxioImager M2 (for immunofluorescence) and analyzed using ImageJ (NIH). Bronchoalveolar lavage Mice were euthanized by cervical dislocation and the lungs were lavaged twice with 0.5ml of a 0.9% saline solution. Bronchoalveolar lavage (BAL) fluid was centrifuged at 500g for 10 minutes to remove cells and debris and the supernatant was collected to quantify total protein leakage into the airway using a BCA assay (Pierce). Cell lysis and damage was analyzed from BAL fluid by measuring lactate dehydrogenase leakage using an LDH assay kit (Sigma). Pulmonary function phenotyping Lung Diffusing Capacity (DF CO ) quantifies the ability of the lung to exchange gas, which is its primary function. Diffusing capacity is simple and quick to measure in humans and mice, and it decreases with nearly all lung pathologies, including viral infections. At the selected time points, a cohort of mice was anesthetized via an IP injection of ketamine–xylazine (100 mg/kg–10 mg/kg), and then an 18-g stub needle was secured in the trachea. 0.8 mL of a gas mixture containing 0.3% neon, 0.3% CO in room air was quickly injected into the lungs, held for 9 s, then quickly withdrawn. This post breathold sample was then injected into a desktop gas chromatograph (Inficon, Micro GC model 3000A) to measure the concentrations of Ne and CO. The DF CO in mice is analogous to the DL CO in humans, and is calculated as 1−(CO9/COc)/(Ne 9 /Ne c ), where subscripts c and 9 refer to the calibration gas injected and the gas from the 9 s exhaled sample. DF CO is thus a dimensionless variable which varies between 0 and 1, and is used to detect the loss and recovery of lung function after the viral infections used in this study [57]. Lung mechanics: After the DF CO is measured, the tracheostomy cannula was then connected to a Flexivent system (Scireq). Ventilation was accomplished at a rate of 150 breaths/minute and a tidal volume of 10 ml/kg with a PEEP of 3 cm H 2 O. A deep inspiration to 30 cmH 2 O was done, and 1 minute later the respiratory resistance (Rrs) and compliance (Crs) were measured [58]. Increased resistance reflects increased difficulty in dynamically moving air into the lung and decreased compliance reflects increased difficulty in expanding the lung parenchyma. Mouse tracheal epithelial cell (mTEC) cultures For mTEC cultures, tracheas were obtained from 7–9 week old C56BL/6 female mice, digested overnight in 0.3% pronase, and enriched by depleting fibroblasts as previously described [59, 60]. The mTECs were cultured at a density of 2.22x105 cells/ml on collagen-coated 24-well transwell plates for 7 days (i.e., until the cultures reached a transepithelial resistance above 1000 Ω· cm2) and apical medium was removed to create an air-liquid interface for 14 days to induce differentiation as described previously [60]. Cells were pre-treated for 24 h with basolateral media containing vehicle (100% ethanol) or 100nM P4 (Sigma), and injured by scratching the cell layer with a 10ul XL pipette tip, or left uninjured, and loose cells were removed by washing with media. Transepithelial cell resistance (TER) was measured prior to injury, immediately after, and every 12h for 48 h by adding 100μl of warm TEC basic media to the apical chamber. New media with vehicle or P4 was added every 24h. Every 12h, basolateral media was sampled and analyzed for AREG expression by ELISA (R&D) according to the manufacturer’s protocol. Cells were harvested in Trizol every 12h and analyzed by RT-PCR as described above. Statistical analyses A power and sample size calculation was used to confirm group sizes for a power of 0.8 and contributes to differential sample sizes for some dependent measures. Repeat measures were analyzed with a multivariate analysis of variance (MANOVA) followed by planned comparisons. Discrete measures were analyzed with T-tests or two-way ANOVA followed by the Tukey method for pairwise multiple comparisons. Survival was analyzed using a Kaplan Meyer survival curve followed by a log-rank test. Mean differences were considered statistically significant if P<0.05.

Supporting Information S1 Table. Cytokine and chemokine concentrations in lung homogenates from ovariectomized female mice treated with placebo (-P4) or progesterone (+P4). https://doi.org/10.1371/journal.ppat.1005840.s001 (DOCX)

Acknowledgments We thank members of the Klein and Pekosz labs for ongoing discussions about these data. We thank Cory Brayton for assisting with the initial histopathological analysis. We thank Alan Scott for feedback on an earlier draft and Matt Craig and Tricia Nilles for technical assistance.

Author Contributions Conceptualization: OJH SLK. Formal analysis: OJH NL MSV. Funding acquisition: AP SLK. Investigation: OJH NL MSV DPR NW. Methodology: AP WM SLK. Project administration: SLK. Resources: AP WM SLK. Supervision: AP WM SLK. Validation: OJH NL MSV DPR NW. Visualization: OJH MSV. Writing – original draft: OJH SLK. Writing – review & editing: OJH NL MSV DPR NW AP WM SLK.