*Article* **Effect of PM2.5 Levels on ED Visits for Respiratory Causes in a Greek Semi-Urban Area**

**Maria Mermiri 1,2,\* , Georgios Mavrovounis <sup>1</sup> , Nikolaos Kanellopoulos <sup>3</sup> , Konstantina Papageorgiou <sup>1</sup> , Michalis Spanos <sup>1</sup> , Georgios Kalantzis <sup>4</sup> , Georgios Saharidis <sup>4</sup> , Konstantinos Gourgoulianis <sup>3</sup> and Ioannis Pantazopoulos <sup>1</sup>**


**Abstract:** Fine particulate matter that have a diameter of <2.5 µm (PM2.5) are an important factor of anthropogenic pollution since they are associated with the development of acute respiratory illnesses. The aim of this prospective study is to examine the correlation between PM2.5 levels in the semi-urban city of Volos and Emergency Department (ED) visits for respiratory causes. ED visits from patients with asthma, pneumonia and upper respiratory infection (URI) were recorded during a one-year period. The 24 h PM2.5 pollution data were collected in a prospective manner by using twelve fully automated air quality monitoring stations. PM2.5 levels exceeded the daily limit during 48.6% of the study period, with the mean PM2.5 concentration being 30.03 <sup>±</sup> 17.47 <sup>µ</sup>g/m<sup>3</sup> . PM2.5 levels were significantly higher during winter. When PM2.5 levels were beyond the daily limit, there was a statistically significant increase in respiratory-related ED visits (1.77 vs. 2.22 visits per day; *p*: 0.018). PM2.5 levels were also statistically significantly related to the number of URI-related ED visits (0.71 vs. 0.99 visits/day; *p* = 0.01). The temperature was negatively correlated with ED visits (r: −0.21; *p* < 0.001) and age was found to be positively correlated with ED visits (r: 0.69; *p* < 0.001), while no statistically significant correlation was found concerning humidity (r: 0.03; *p* = 0.58). In conclusion, PM2.5 levels had a significant effect on ED visits for respiratory causes in the city of Volos.

**Keywords:** PM2.5; air pollution; respiratory diseases; asthma; pneumonia; upper respiratory infections; emergency department

#### **1. Introduction**

Anthropogenic air pollution is a major cause of environmental pollution, which can have detrimental effects on human health [1]. Fine particle matter 2.5 (PM2.5) are fine particles that have an aerodynamic diameter of less than 2.5 µm, which are produced mainly by wood and fuel combustion [2]. Because of their small diameters, they are able to infiltrate through the nose into the lower respiratory system, accumulating in respiratory bronchioles [2]. Alveolar damage may consequently occur due to the production of free radicals, imbalanced intracellular homeostasis and inflammation [2].

High levels of PM2.5 have been previously associated with several respiratory diseases, as well as increased morbidity and mortality due to respiratory causes [3]. In a metaanalysis of 16 studies, short-term exposure to increased PM2.5 levels were shown to precipitate asthma attacks leading to increased hospitalizations in patients suffering from severe respiratory disorders [4]. Moreover, a prospective study of 431 patients suffering

**Citation:** Mermiri, M.; Mavrovounis, G.; Kanellopoulos, N.; Papageorgiou, K.; Spanos, M.; Kalantzis, G.; Saharidis, G.; Gourgoulianis, K.; Pantazopoulos, I. Effect of PM2.5 Levels on ED Visits for Respiratory Causes in a Greek Semi-Urban Area. *J. Pers. Med.* **2022**, *12*, 1849. https:// doi.org/10.3390/jpm12111849

Academic Editor: Eumorfia Kondili

Received: 30 July 2022 Accepted: 31 October 2022 Published: 5 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

from Chronic Obstructive Pulmonary Disease (COPD) in a highly polluted city of Italy showcased that high PM10 and PM2.5 levels may precipitate COPD exacerbations and increase hospital admissions in these patients. Furthermore, several prospective studies and a meta-analysis by Kim et al. proved that elevated PM2.5 levels have been associated with a surge in Emergency Department (ED) visits for respiratory causes, such as asthma attacks and respiratory infections [4–6].

While the connection between PM2.5 pollution and respiratory diseases has been extensively researched, there is a lack of studies pertaining to the relationship between air pollution and health in rural or semi-urban areas. Although these areas may have lower exposure to industrial and motor vehicle pollution [7], studies have shown that several non-urban regions are greatly exposed to pollution produced by agriculture, coal burning and mining and wood burning [8,9]. The semi-urban city of Volos is a medium-size coastal city in Greece, where high levels of PM10 and PM2.5 pollution have been previously recorded [10,11]. Moustris et al. studied the effect of PM10 pollution in hospital admissions for respiratory diseases during a five-year period in Volos [10]. Their results showcased that increased PM10 levels were associated with an 25% increase in hospital admissions. Later, our team in a similarly designed study studied the effect of PM2.5 levels in pediatric ED visits for respiratory causes during a one-year period [11]. Likewise, elevated PM2.5 levels resulted in an increase in pediatric ED visits for respiratory causes.

The aim of the present study was to investigate the relationship between daily PM2.5 levels in the semi-urban Greek city of Volos and the number of adult ED visits for respiratory causes.

#### **2. Materials and Methods**

#### *2.1. Study Design and Population*

This study retrospectively analysed a prospectively collected database. The study was approved by the local scientific committee (2/7-2-2019). The methodology has been previously described in detail for our pediatric ED visits study [11].

Data were collected for adult patients visiting the ED of Volos with respiratory symptoms, including asthma, pneumonia, COPD and upper respiratory tract infection (URI) related visits between 1 March 2019 and 29 February 2020. We used the following inclusion criteria: (1) residents of Volos city, (2) those who visited the adult ED and (3) those with respiratory conditions (URI, COPD exacerbation, asthma exacerbation and pneumonia). The following data were collected for all patients: age, gender, address of residence, main complaint/diagnosis and date of visit.

#### *2.2. Network of Sensors*

The GreenYourAir research group established a fully automated network for monitoring air pollution in the city of Volos. Twelve stations located throughout the central and greater Volos areas collected the data prospectively during the entire study period. The locations were chosen based on a mathematical formula and an optimization model developed by the GreenYourAir team that parcellated the city into smaller areas. Briefly, the team divided the city into five distinct areas, namely the commercial and recreational zone; the industrial zone; and the high-density, medium-density and low-density residential zones, while the traffic jam of the city was divided into three categories (high, medium and low). Furthermore, additional information regarding the city was taken into consideration, such as the geographical and geomorphological characteristics of the area, the commercial port, the passenger port, the urban and intercity bus stations, the train station, the main roads, the recreational parks, the sport facilities, the schools and other academic institutions and two big industrial plants located just outside the city (one cement company and one petroleum company). It should be mentioned that the residents of the city use oil, natural gas and fireplaces for heating during the winter. The exact location of the sensors throughout the city of Volos is presented in Figure 1.

the sensors throughout the city of Volos is presented in Figure 1.

company and one petroleum company). It should be mentioned that the residents of the city use oil, natural gas and fireplaces for heating during the winter. The exact location of

**Figure 1.** Map of the city of Volos presenting the location of sensors. Markers indicate the location of the sensors. **Figure 1.** Map of the city of Volos presenting the location of sensors. Markers indicate the location of the sensors.

The devices that were used for monitoring (GreenYourAir Device 1178/PM2.5) used light-scattering methods for data collection, as described previously in the literature [12,13]. The devices consist of a sensor, an expansion shield and an Arduino YUN rev. 2. The sensor collects data regarding PM2.5 concentration, temperature and humidity in the area. The collection of data was performed automatically every second for the entire 24 h The devices that were used for monitoring (GreenYourAir Device 1178/PM2.5) used light-scattering methods for data collection, as described previously in the literature [12,13]. The devices consist of a sensor, an expansion shield and an Arduino YUN rev. 2. The sensor collects data regarding PM2.5 concentration, temperature and humidity in the area. The collection of data was performed automatically every second for the entire 24 h of the day. The programming language used for the devices was C++.

of the day. The programming language used for the devices was C++. A two-phase calibration methodology was implemented to check the accuracy of the devices. During the first stage development and testing period, the sensors were validated in laboratory conditions using reference equipment that followed EU standards EN A two-phase calibration methodology was implemented to check the accuracy of the devices. During the first stage development and testing period, the sensors were validated in laboratory conditions using reference equipment that followed EU standards EN 14907:2005 (gravimetric device with filters that collects PM2.5). Later, during the second stage, the sensors were validated in real-life conditions.

14907:2005 (gravimetric device with filters that collects PM2.5). Later, during the second stage, the sensors were validated in real-life conditions. The network created started operating on 1 March 2019 and is still working 24/7 at The network created started operating on 1 March 2019 and is still working 24/7 at the time of writing this manuscript. The real-time data for the city of Volos can be found at http://greenyourair.org/ (accessed on 30 October 2022).

#### the time of writing this manuscript. The real-time data for the city of Volos can be found at http://greenyourair.org/ (accessed on 3 November 2022). *2.3. Statistical Analysis*

*2.3. Statistical Analysis* The independent samples t-test and analysis of variance were used for the between groups comparisons of continuous variables as appropriate. The chi-square test was used to identify possible relationships between categorical variables. We used regression analysis and Pearson's correlation in order to describe the relationship between the number of ED visits and temperature, humidity, age and PM2.5 levels. The significance for all tests The independent samples t-test and analysis of variance were used for the between groups comparisons of continuous variables as appropriate. The chi-square test was used to identify possible relationships between categorical variables. We used regression analysis and Pearson's correlation in order to describe the relationship between the number of ED visits and temperature, humidity, age and PM2.5 levels. The significance for all tests was set at *p* values < 0.05 and all tests were two-tailed. The SPSS statistical package was used for all statistical analyses (IBM Corp. Released 2016. IBM SPSS Statistics for Windows, Version 24.0. Armonk, NY, USA; IBM Corp).

#### was set at *p* values < 0.05 and all tests were two-tailed. The SPSS statistical package was **3. Results**

#### used for all statistical analyses (IBM Corp. Released 2016. IBM SPSS Statistics for Win-*3.1. Patient Characteristics*

dows, Version 24.0. Armonk, NY, USA; IBM Corp). During our study period, a total of 728 patients visited the adult ED for respiratory causes. The male to female ratio was 1.05 (373 male and 355 female patients), while the mean age for male patients was 64.50 ± 19.69, and for female patients, it was 61.43 ± 20.52.

A total of 310 patients (mean age (standard deviation): 66.35 ± 20.77; male gender: 166 (54%)) were diagnosed with an URI, 202 patients were diagnosed with a lower respiratory tract infection/pneumonia (mean age (standard deviation): 52.41 ± 20.70; male gender: 106(52%)), 53 patients were diagnosed with asthma exacerbation (mean age (standard deviation): 50.39 ± 20.05; male gender: 15(28%)) and 163 patients were diagnosed with COPD exacerbation (mean age (standard deviation): 69.49 ± 12.20; male gender: 85(52%)). der: 106(52%)), 53 patients were diagnosed with asthma exacerbation (mean age (standard deviation): 50.39 ± 20.05; male gender: 15(28%)) and 163 patients were diagnosed with COPD exacerbation (mean age (standard deviation): 69.49 ± 12.20; male gender: 85(52%)). *3.2. PM2.5 Levels* In the city of Volos, the mean annual PM2.5 concentration during the study period

During our study period, a total of 728 patients visited the adult ED for respiratory causes. The male to female ratio was 1.05 (373 male and 355 female patients), while the mean age for male patients was 64.50 ± 19.69, and for female patients, it was 61.43 ± 20.52. A total of 310 patients (mean age (standard deviation): 66.35 ± 20.77; male gender: 166 (54%)) were diagnosed with an URI, 202 patients were diagnosed with a lower respiratory tract infection/pneumonia (mean age (standard deviation): 52.41 ± 20.70; male gen-

*J. Pers. Med.* **2022**, *12*, x FOR PEER REVIEW 4 of 10

#### *3.2. PM2.5 Levels* was calculated to be 30.03 ± 17.47 μg/m3 compared to WHO's yearly limit of 10 μg/m3.

**3. Results**

*3.1. Patient Characteristics*

In the city of Volos, the mean annual PM2.5 concentration during the study period was calculated to be 30.03 <sup>±</sup> 17.47 <sup>µ</sup>g/m<sup>3</sup> compared to WHO's yearly limit of 10 µg/m<sup>3</sup> . Overall, the suggested daily limit of PM2.5 (25 µg/m<sup>3</sup> ) was exceeded for 178 days, which was 48.60% of the study period. The recorded levels of PM2.5 were found to be higher in winter when compared with summer (mean difference: 25.64, *p*: < 0.001), autumn (mean difference: 20.20, *p*: < 0.001) and spring (mean difference: 17.71, *p*: < 0.001). Overall, the suggested daily limit of PM2.5 (25 μg/m3) was exceeded for 178 days, which was 48.60% of the study period. The recorded levels of PM2.5 were found to be higher in winter when compared with summer (mean difference: 25.64, *p*: < 0.001), autumn (mean difference: 20.20, *p*: < 0.001) and spring (mean difference: 17.71, *p*: < 0.001). *3.3. PM2.5 levels, Humidity, Temperature, Age and ED Visits*

#### *3.3. PM2.5 Levels, Humidity, Temperature, Age and ED Visits* The mean number of daily ED visits for respiratory causes was 1.99 ± 1.81. The num-

The mean number of daily ED visits for respiratory causes was 1.99 ± 1.81. The number of monthly ED alongside the corresponding monthly PM2.5 levels are presented in Figure 2. As shown in Table 1, a 25.42% increase in daily ED visits for all respiratory causes was identified when PM2.5 levels exceeded the daily limit (1.77 vs. 2.22 visits per day; *p*: 0.018). Further analyses performed by season (Table 2) illustrated that the increase in ED visits was more pronounced during winter (34.08%) and autumn (29.40%), although the difference was non-statistically significant. Moreover, there was statistically significant elevation in ED visits when PM2.5 levels of the previous day were higher than the proposed limit 25 µg/m<sup>3</sup> (*p* = 0.018). ber of monthly ED alongside the corresponding monthly PM2.5 levels are presented in Figure 2. As shown in Table 1, a 25.42% increase in daily ED visits for all respiratory causes was identified when PM2.5 levels exceeded the daily limit (1.77 vs. 2.22 visits per day; *p*: 0.018). Further analyses performed by season (Table 2) illustrated that the increase in ED visits was more pronounced during winter (34.08%) and autumn (29.40%), although the difference was non-statistically significant. Moreover, there was statistically significant elevation in ED visits when PM2.5 levels of the previous day were higher than the proposed limit 25 μg/m3 (*p* = 0.018).

Additionally, we identified that temperature was negatively correlated with ED visits (r: −0.21; *p* < 0.001), while humidity did not exhibit any statistically significant correlation (r: 0.03; *p* = 0.58). Finally, age was found to be positively correlated with ED visits (r: 0.69; *p* < 0.001). Additionally, we identified that temperature was negatively correlated with ED visits (r: −0.21; *p* < 0.001), while humidity did not exhibit any statistically significant correlation (r: 0.03; *p* = 0.58). Finally, age was found to be positively correlated with ED visits (r: 0.69; *p* < 0.001).

**Figure 2. Figure 2.**  Monthly PM2.5 levels and ED visits. Monthly PM2.5 levels and ED visits.


**Table 1.** Table presenting the number of ED visits in relation to daily PM2.5 levels.

Abbreviations: ED: emergency department; SD: standard deviation; URI: upper respiratory tract infection; COPD: Chronic Obstructive Pulmonary Disease.

**Table 2.** Table presenting the number of ED visits in relation to mean PM2.5 levels of each season.


Abbreviations: ED: emergency department; SD: standard deviation.

#### *3.4. Specific Conditions and PM2.5 Levels*

A statistically significant increase in ED visits for URI was noted during the days that PM2.5 levels exceeded the limit of 25 µg/m<sup>3</sup> , when compared to the days when PM2.5 levels were below 25 µg/m<sup>3</sup> (0.71 vs. 0.99 visits/day; *p* = 0.01). Table 1 presents the comparisons for all studied conditions. No statistically significant differences were identified when a further analysis was performed based on the season (Supplementary File S1). Finally, no statistically significant differences in mean ED visits were observed for all studied conditions (URI: *p* = 0.05; Pneumonia: *p* = 0.42; Asthma: *p* = 0.28; COPD: *p* = 0.47) when comparing males and females.

In regression analysis, a linear correlation (r square: 0.022; *p* < 0.001) was noted between the levels of PM2.5 and the total number of daily ED visits. This is described by the following equation: total number of ED visits = 1.524 + 0.015 × PM2.5 levels (Figure 3). When we included temperature and humidity in the model's parameters, the model was still statistically significant (r square: 0.047; *p* < 0.001) although the only parameter with statistical significance was temperature.

**Figure 3.** Correlation between daily PM2.5 levels and daily ED visits. **Figure 3.** Correlation between daily PM2.5 levels and daily ED visits.

#### **4. Discussion**

*4.* **Discussion** In our study, PM2.5 levels exceeded the daily limit of 10 μg/m3 during 48.6% of the study period, with the mean PM2.5 concentration being 30.03 ± 17.47 μg/m<sup>3</sup> . PM2.5 levels were higher during the winter compared to autumn, spring, and summer. ED visits were significantly higher on days when PM2.5 concentrations exceeded the daily limit, or the day after. Although age and temperature had a significant correlation with ED visits, humidity did not play a role in the number of daily ED visits. Moreover, high PM2.5 levels In our study, PM2.5 levels exceeded the daily limit of 10 µg/m<sup>3</sup> during 48.6% of the study period, with the mean PM2.5 concentration being 30.03 <sup>±</sup> 17.47 <sup>µ</sup>g/m<sup>3</sup> . PM2.5 levels were higher during the winter compared to autumn, spring, and summer. ED visits were significantly higher on days when PM2.5 concentrations exceeded the daily limit, or the day after. Although age and temperature had a significant correlation with ED visits, humidity did not play a role in the number of daily ED visits. Moreover, high PM2.5 levels were associated with an increase in URI-related ED visits.

were associated with an increase in URI-related ED visits. Air pollution has become increasingly prevalent during the last decades, since many global areas are exposed daily to high levels of air pollutants [14]. While large metropolises suffer greatly [15], semi-urban and rural areas can also be affected [8]. Research pertaining to the relationship between air pollution and human health in rural and semi-ur-Air pollution has become increasingly prevalent during the last decades, since many global areas are exposed daily to high levels of air pollutants [14]. While large metropolises suffer greatly [15], semi-urban and rural areas can also be affected [8]. Research pertaining to the relationship between air pollution and human health in rural and semi-urban areas may further highlight the importance of air pollutants on respiratory health.

ban areas may further highlight the importance of air pollutants on respiratory health. Despite the fact that the city of Volos is a semi-urban area, high levels of air pollutants have been previously recorded [10,11]. In accordance with our study, Moustris et al. [10] reported elevated levels of PM10 pollution in the city of Volos, with PM10 levels regularly exceeding the daily and annual proposed limits by WHO. In the study by Moustris et al., an increase in the annual PM10 concentration in the city of Volos resulted in an increase in the annual hospital admissions for respiratory diseases. However, in our study, we researched the relationship between daily PM2.5 concentrations and ED visits for respira-Despite the fact that the city of Volos is a semi-urban area, high levels of air pollutants have been previously recorded [10,11]. In accordance with our study, Moustris et al. [10] reported elevated levels of PM10 pollution in the city of Volos, with PM10 levels regularly exceeding the daily and annual proposed limits by WHO. In the study by Moustris et al., an increase in the annual PM10 concentration in the city of Volos resulted in an increase in the annual hospital admissions for respiratory diseases. However, in our study, we researched the relationship between daily PM2.5 concentrations and ED visits for respiratory causes, which further highlights the direct effect of PM2.5 pollution and respiratory health.

tory causes, which further highlights the direct effect of PM2.5 pollution and respiratory health. PM2.5 production is mainly anthropogenic [14]. Fine particle matter is produced primarily through fuel and wood combustion, mainly deriving from vehicle and biofuel emissions [16]. A meta-analysis of PM source apportionment in Europe revealed that traffic, as well as wood burning during the cold months, were the most important factors in PM production [16]. In the last decade, the number of Greek households using wood burning as a means of heating during the winter rapidly increased due to the high petrol prices and economic crisis [17]. This increase in wood burning has subsequently led to an increase in fine PM levels, which are significantly higher during the cold months [17]. Likewise, the present study indicates that PM2.5 levels in the city of Volos are higher during winter compared to other seasons. This relationship may indicate that the production of PM2.5 production is mainly anthropogenic [14]. Fine particle matter is produced primarily through fuel and wood combustion, mainly deriving from vehicle and biofuel emissions [16]. A meta-analysis of PM source apportionment in Europe revealed that traffic, as well as wood burning during the cold months, were the most important factors in PM production [16]. In the last decade, the number of Greek households using wood burning as a means of heating during the winter rapidly increased due to the high petrol prices and economic crisis [17]. This increase in wood burning has subsequently led to an increase in fine PM levels, which are significantly higher during the cold months [17]. Likewise, the present study indicates that PM2.5 levels in the city of Volos are higher during winter compared to other seasons. This relationship may indicate that the production of PM2.5 by wood burning may be a significant component of environmental pollution in semi-urban or rural areas, where pollutant production by industrial processes is less prominent.

Approximately 4.2 premature million deaths annually can be attributed to PM2.5 pollution, placing fine particle pollution as the fifth most common cause of death worldwide [18]. Exposure to elevated PM2.5 levels has been associated with increased Out-of-Hospital Cardiac Arrests (OHCAs), as well as a variety of cardiovascular and respiratory diseases [19–21]. Interestingly, a meta-analysis by Atkinson et al. [20] demonstrated that PM2.5 pollution is more strongly associated with mortality due to respiratory causes [20]. Indeed, high PM2.5 levels are associated with increased rates of respiratory infections and asthma or COPD exacerbations, leading to an increase in ED visits for respiratory causes [22,23].

According to our findings, in the semi-urban city of Volos, there was a statistically significant association between PM2.5 levels and ED visits for respiratory causes. These findings are in accordance with two similar studies conducted in the city of Volos, which demonstrated that fine particulate matter pollution is associated with increased hospital admissions for respiratory causes in both adult and pediatric patients [10,11]. As previously mentioned, high levels of PM2.5 can precipitate a variety of respiratory diseases, such as respiratory infections and asthma and COPD exacerbations, as well as increased hospital admissions and mortality due to respiratory causes [20,22]. Specifically, our research team, using the model developed by the GreenYourAir research group, discovered that pediatric ED visits in the city of Volos were linearly correlated with PM2.5 levels [11]. In our study, the same research group discovered that a similar relationship exists in adult patients. Furthermore, our results revealed a significant correlation between age and ED visits for respiratory causes. As showcased in previous studies, the effect of PM2.5 on respiratory diseases seems to be more pronounced in children and adults [7,24] According to previous studies, the effect of PM2.5 on respiratory-related conditions is more significant in adults more than 65 years old [7].

Several meteorological factors have been shown to influence the concentration of PM2.5 and its effect on human health [25,26]. In our study, a decrease in the mean daily temperature resulted in an increase in daily ED visits while humidity did not have a statistically significant correlation with ED visits. Similarly, a study by Wang et al. in 28 Chinese cities showcased that low temperatures during winter were correlated with higher PM2.5 concentrations [25]. The researchers hypothesized that this effect may be due to the increased coal and wood burning during the cold months. Moreover, low temperature has been associated with an increased susceptibility to URIs [27], which may increase respiratory-related ED visits. However, a study in Lima, Peru, showcased that the effect of PM2.5 concentrations in respiratory and cardiac mortality was more pronounced when the mean daily temperature was higher than 23.8 ◦C [28]. A similar relationship between PM10 and respiratory disease was found in a systematic review and meta-analysis by Chen et al. [29], which found that the effect of air pollution on respiratory diseases is more significant when the temperature is higher. These conflicting results prove that more research is necessary in order to accurately understand the complicated effects of meteorological factors in fine particulate matter concentrations and their effects on human health. Moreover, while our results did not reveal a significant correlation between humidity and ED visits, it is important to note that several researchers proved that high relative humidity may be accompanied by higher PM2.5 and PM10 concentrations, which may have a negative effect on respiratory-related ED visits [30,31].

In our study, there was a statistically significant association between URI-related ED visits and PM2.5 levels. The association between PM2.5 levels and respiratory infections has been proved by numerous studies, which showcase that increased PM2.5 levels lead to increased ED visits and hospital admissions for upper and lower respiratory infections [7,32]. The study by our research team in the city of Volos, also indicated that high daily PM2.5 levels are correlated with increased pediatric ED visits for URI [11]. This effect has been attributed to increased susceptibility to infections [33] based on experimental animal models [34,35]. While the exact mechanism remains unknown, it is speculated that PM2.5 impairs the host's defense of the respiratory system by altering epithelial cell functions

and immune cell activity [33]. This effect is more pronounced in pediatric and elderly patients [33].

The association between PM2.5 pollution and chronic respiratory diseases has been well documented. Both PM2.5 and PM10 may cause COPD exacerbations, leading to more ED visits for respiratory causes and increased morbidity and mortality in COPD patients [22,36]. Interestingly, it has been demonstrated that PM2.5 exposure may cause chronic respiratory dysfunction, creating emphysematous lesions and chronic inflammation, which in turn may lead to COPD developments [36]. Moreover, PM2.5 pollution can aggravate the effects of smoking on lung function, increasing the likelihood of COPD [36]. In contrast to the existing literature, no statistically significant association between PM2.5 levels and COPD association was noted in our study. However, this discrepancy may be attributed to the small number of patients presenting to the ED with respiratory symptoms due to COPD exacerbation.

PM2.5 pollution may also worsen asthma symptoms, leading to increased ED visits and hospital admissions for asthma exacerbations [4,37,38]. A meta-analysis by Fan et al. demonstrated that asthma-related ED visits increased proportionally to PM2.5 levels [4]. The effect was more pronounced in pediatric patients. Moreover, asthma exacerbations are most likely to occur during spring possibly due to the prevalence of allergens, such as pollen [37]. The association between PM2.5 pollution and asthma can be attributed to the increased inflammation of airway epithelial cells and the increased secretion of inflammatory cytokines [39,40]. Our study did not reveal a statistically significant association between PM2.5 levels and asthma-related ED visits. Moreover, our team in a similar study in pediatric patients did not discover a statistically significant relationship between asthmarelated ED visits and PM2.5 levels. Similarly to COPD cases, this could be explained by the small number of asthma-related ED visits during our study period.

#### *Limitations*

Our study has some limitations that should be acknowledged. Firstly, is it a singlecenter study that collected data from a single ED; thus, our results should be interpreted with caution. Moreover, the diagnoses were made in an emergency setting; therefore, there may have been some misdiagnoses. It is also important to note that our analysis was conducted based on the mean PM2.5 levels of each day and may not accurately reflect patient exposure, since no regional analysis was conducted.

#### **5. Conclusions**

In our study, high PM2.5 levels were associated with an increase in adult ED visits for respiratory causes. PM2.5 pollution was statistically significantly related to the number of URI-related visits but not COPD or asthma-related visits. Moreover, low temperatures and increased age increased ED visits for respiratory causes. While the association between fine particle pollution and respiratory illnesses has been well documented, more studies are necessary in order to ascertain the pathophysiological mechanisms leading to respiratory dysfunction, as well as individual factors that may predispose certain patients to PM2.5 related respiratory symptoms.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jpm12111849/s1.

**Author Contributions:** Conceptualization, N.K., G.S., G.K. and K.G.; methodology, N.K., G.S., G.K. and K.G.; software, G.S. and G.K.; formal analysis, G.S., GK. and M.S.; data curation, G.S., G.K., M.S. and K.P.; writing—original draft preparation, M.M., G.M. and K.P.; writing—review and editing, N.K., I.P., K.G. and M.M.; visualization, G.S. and G.K.; supervision, I.P. and K.G.; project ad-ministration, I.P. and K.G.; funding acquisition, K.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the University of Thessaly (2/7-2-2019).

**Informed Consent Statement:** Patient consent was waived due to the retrospective nature of the data analysis. Patient anonymity and confidentiality were maintained through all stages of the data analysis process.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author (MM) upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

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**Ourania S. Kotsiou 1,\* , Ioannis Pantazopoulos <sup>2</sup> , Georgios Mavrovounis <sup>3</sup> , Konstantinos Marsitopoulos <sup>3</sup> , Konstantinos Tourlakopoulos <sup>3</sup> , Paraskevi Kirgou <sup>3</sup> , Zoe Daniil <sup>3</sup> and Konstantinos I. Gourgoulianis <sup>3</sup>**


**\*** Correspondence: raniakotsiou@gmail.com

**Abstract:** Introduction: The most clinically useful concept in asthma is based on the intensity of treatment required to achieve good asthma control. Biomarkers to guide therapy are needed. Aims: To investigate the role of circulating levels of soluble urokinase plasminogen activator receptor suPAR as a marker for asthma severity. Methods: We recruited patients evaluated at the Asthma Clinic, University of Thessaly, Greece. Asthma severity and control were defined according to the GINA strategy and Asthma Contro Test (ACT). Anthropometrics, spirometry, fractional exhaled nitric oxide (FeNO), suPAR, blood cell count, c-reactive protein (CRP), and analyses of kidney and liver function were obtained. Patients with a history of inflammatory, infectious, or malignant disease or other lung disease, more than 5 pack years of smoking history, or corticosteroid therapy were excluded. Results: We evaluated 74 asthma patients (69% female, mean age 57 ± 17 years, mean body mass index (BMI) <sup>29</sup> <sup>±</sup> 6 kg/m<sup>2</sup> ). In total, 24%, 13%, 6%, 5%, 29% and 23% of the participants had mild well-controlled, mild uncontrolled, moderate well-controlled, moderate uncontrolled, severe well-controlled, and severe uncontrolled asthma, respectively. Overall, 67% had T2-high asthma, 26% received biologics (15% and 85% received omalizumab and mepolizumab, respectively), and 34% had persistent airway obstruction. suPAR levels were significantly lower in asthmatics with moderate uncontrolled asthma than in patients with severe uncontrolled asthma without (2.1 ± 0.4 vs. 3.3 ± 0.7 ng/mL, *p* = 0.023) or with biologics (2.1 ± 0.4 vs. 3.6 ± 0.8 ng/mL, *p* = 0.029). No correlations were found between suPAR levels and age, BMI, T2 biomarkers, CRP, or spirometric parameters. Conclusions: suPAR levels were higher in asthmatics with severe disease than in those with moderate uncontrolled asthma.

**Keywords:** asthma; control; severity; urokinase plasminogen activator receptor

### **1. Introduction**

The soluble urokinase plasminogen activator receptor (suPAR) is the circulating form of the cell surface receptor urokinase plasminogen activator receptor (uPAR) (CD87), which is expressed by a plethora of cells ranging from mono- and lymphocytes to endothelial and smooth muscle cells [1]. suPAR is a novel biomarker playing an important role in many physiological and pathological processes, including endothelial dysfunction, thrombosis [2], inflammation [1–4], chemotaxis [1,2], tissue remodeling [1–4], and tumorigenesis [1,2]. suPAR's high stability in plasma samples makes it an ideal candidate biomarker in patients with inflammatory, infectious, and malignant diseases [1–7]. Recently, a position statement on the prognostic role of suPAR in the screening of patients admitted to the emergency department was issued by the Hellenic Sepsis Study Group.

A remarkable role for suPAR serum levels has been demonstrated in airway diseases. It has been reported that suPAR can be used to evaluate stable chronic obstructive disease (COPD) [3] as a predictor of acute exacerbation and in monitoring response to treatment [3]. Moreover, suPAR has a significant role in increased systemic inflammation associated with coexisting COPD and bronchiectasis [4].

**Citation:** Kotsiou, O.S.; Pantazopoulos, I.; Mavrovounis, G.; Marsitopoulos, K.; Tourlakopoulos, K.; Kirgou, P.; Daniil, Z.; Gourgoulianis, K.I. Serum Levels of Urokinase Plasminogen Activator Receptor (suPAR) Discriminate Moderate Uncontrolled from Severe Asthma. *J. Pers. Med.* **2022**, *12*, 1776. https://doi.org/10.3390/jpm12111776

Academic Editor: Bruno Mégarbané

Received: 21 September 2022 Accepted: 24 October 2022 Published: 28 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Evidence has shown that airway inflammation might spread into the circulatory system and cause systemic inflammatory injuries. In that context, emerging data support asthma associated with chronic low-grade systemic inflammation, a prothrombotic state, and premature atherosclerosis, even in clinically stable asthma patients [5]. Asthma is also characterized by endothelial dysfunction related to airway obstruction [5]. There is evidence of an abnormal amount of endothelial tissue in asthma and that this tissue and its progenitor cells behave in a dysfunctional manner [6]. Sputum, biopsy and serum suPAR levels were elevated in stable asthma patients compared to controls [7]. Moreover, in asthmatic patients, high suPAR indicated impaired lung function and was shown to correlate with airway resistance [1]. However, no data on the potential role of circulating suPAR as a marker for asthma severity and prognosis according to severity have been reported so far.

Patients with severe asthma are at a particularly high risk of exacerbations, hospitalization and death and have severely impaired quality of life. On the other hand, patients with mild asthma (the silent majority of asthmatics) account for the majority of the morbidity and healthcare resource utilization associated with asthma [1,8].

The aim of this study was to investigate the effectiveness of suPAR as an indicator of the severity of asthma.

#### **2. Methods**

#### *2.1. Study Design*

The recruitment period of the study lasted 6 months. Detailed lung function tests were performed in severe asthmatic patients and control groups in the following order: measurement of the fraction of exhaled nitric oxide (FeNO and spirometry) [9]. Severe asthmatics and controls had a set of standard blood tests analyzed, including suPAR, blood cell count (white blood cell count, eosinophils %, absolute eosinophil count), Creactive protein (CRP), electrolytes (sodium, potassium), and analyses of the kidney (urea, creatinine) and liver function (aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transpeptidase (γGT), and alkaline phospatase (ALP)) at baseline. The Ethics Committee of the University of Thessaly approved the protocol. Written informed consent was obtained.

#### *2.2. Participants*

All patients were well-defined regarding asthma severity as they were managed by an experienced pulmonologist in the external Unit of Asthma of the University of Thessaly in Greece for at least one year prior to recruitment. Exclusion criteria were: any history of acute (within four weeks of recruitment) or chronic inflammatory, infectious or malignant disease, COPD and/or other relevant lung diseases causing alternating impairment in lung function, current smoking or more than 5 pack years of smoking history, hypertension, diabetes mellitus, angiopathy, renal disorder, or corticosteroid therapy.

#### *2.3. Assessment of the Severity of Asthma*

The severity of asthma was assessed according to the level of treatment required to control symptoms and exacerbations according to GINA 2022 [8]. Mild asthma was defined as asthma that was well controlled while treated with as-needed inhaled corticosteroids (ICS)/formoterol or with low-dose ICSs, plus an as-needed short-acting bronchodilator (SABA) [8]. Moderate asthma was defined as asthma that was well-controlled with a low- or medium-dose ICS-long-acting bronchodilator (LABA) (with step 3 or step 4 treatment) [8]. Severe asthma was defined as asthma that required high-dose ICS/LABA to prevent it from becoming uncontrolled or asthma that remained uncontrolled despite this treatment [8].

#### *2.4. Asthma Control Evaluation*

Asthma control was assessed according to the level of symptom control [8]. Symptom control was determined using the Asthma Control Test (ACT) [10] and discriminated as well-controlled or uncontrolled [8]. In the ACT, scores range from 5–25. Scores of 20–25 were classified as controlled and 5–19 as not well-controlled [10].

#### *2.5. Fraction of Exhaled Nitric Oxide (FeNO)*

The FeNO (MEDISOFT, MEDICAL GRAPHICS CORP, Minnesota, USA) was performed according to recommendations [11]. Recommended cut-off values for normal FeNO levels were <25 parts per billion (ppb) [11].

#### *2.6. Spirometry*

Lung function was measured by means of an electronic spirometer (Spirolab FCC ID: TUK-MIR045) according to the American Thoracic Society (ATS) guidelines [12]. Persistent airflow limitation was defined as forced expiratory volume in the first second (FEV1)/forced vital capacity (FVC) ratio consistently < 70% despite irreversibility in asthma expressed as an increase in FEV1 ≥ 12% and 200 mL [8].

#### *2.7. Blood Sample Collection*

Peripheral venous blood samples were collected in sterile, pro-coagulation tubes and centrifuged immediately; the resulting serum samples were stored at −80 ◦C until analysis. Plasma suPAR levels were measured using the suPARnostic AUTO Flex ELISA kit (ViroGates A/S, Birkerød, Denmark) as described in detail previously [13]. The suPARnostic ELISA measures the full-length suPAR molecule (D1D2D3) and the cleaved suPAR molecule (D2D3). CRP was measured using a COBAS 6000 analyzer (Roche Diagnostics, Mannheim, Germany).

#### *2.8. Statistical Analyses*

The Pearson correlation method was used for correlation analysis between pairs of continuous variables. To identify differences between two independent groups, an unpaired t-test was used. Parametric data comparing three or more groups were analyzed with one-way ANOVA and Tukey's multiple comparisons test, while non-parametric data were analyzed with the Kruskal–Wallis test and Dunn's multiple comparison test. Pearson's chisquared test was used to determine whether there was a statistically significant difference between frequencies. A result was considered statistically significant when the *p*-value was <0.05. Data were analyzed and visualized using SPSS Statistics v. 23 (Armonk, NY, USA, IBM Corp.) and GraphPad Prism 8.

#### **3. Results**

We evaluated 74 asthma patients. A total of 69% of them were female. The mean age of the population was 57 <sup>±</sup> 17 years. The mean body mass index (BMI) was 29 <sup>±</sup> 6 kg/m<sup>2</sup> (Table 1).

Overall, 24%, 13%, 6%, 5%, 29% and 23% of the participants had mild well-controlled, mild uncontrolled, moderate well-controlled, moderate uncontrolled, severe well-controlled, and severe uncontrolled asthma, respectively.

Furthermore, 67% of the population had T2-high asthma according to the measured T2 high biomarkers (blood eosinophil count ≥ 150 cells/µL and FeNO ≥ 20 ppb). Overall, 26% received biologics (15% and 85% received omalizumab and mepolizumab, respectively), while 34% had persistent airway obstruction. The demographic, clinical and spirometric characteristics of the study population are presented in Table 1. Females had significantly less symptom control than males (Table 1).

Abbreviations: BMI, body mass index; FeNO, fraction of exhaled nitric oxide; FEV1, forced expiratory volume in the first second; FVC, forced vital capacity. No significant differences regarding the measured laboratory parameters (white blood cell count, CRP, sodium, potassium, urea, creatinine, AST, ALT, γGT, and ALP were detected among genders. The comparison of serum suPAR levels among groups of asthmatics is presented in Table 1.

BMI (kg/m<sup>2</sup>


**Table 1.** Demographic, clinical and spirometric characteristics of the asthmatics (*n* = 74). Age (years) 57 ± 17 52 ± 18 59 ± 16 0.089 ) 29 ± 6 28 ± 6 29 ± 6 0.758

**Table 1.** Demographic, clinical and spirometric characteristics of the asthmatics (*n* = 74).

26% received biologics (15% and 85% received omalizumab and mepolizumab, respectively), while 34% had persistent airway obstruction. The demographic, clinical and spirometric characteristics of the study population are presented in Table 1. Females had sig-

**1131(***<sup>n</sup>* **= 52)** *<sup>p</sup>***-Value**

*J. Pers. Med.* **2022**, *12*, x FOR PEER REVIEW 4 of 9

nificantly less symptom control than males (Table 1).

Note: Data are expressed as mean ± SD or as frequencies (percentages). differences regarding the measured laboratory parameters (white blood cell count, CRP,

suPAR levels were significantly higher in asthmatics with severe uncontrolled asthma not receiving biologics than in patients with moderate uncontrolled asthma (3.3 ± 0.7 vs. 2.1 ± 0.4 ng/mL, *p* = 0.023) (Figure 1). Moreover, suPAR levels were significantly lower in asthmatics with moderate uncontrolled asthma than in patients with severe uncontrolled asthma without (2.1 ± 0.4 vs. 3.3 ± 0.7 ng/mL, *p* = 0.023) or with biologics (2.1 ± 0.4 vs. 3.6 ± 0.8 ng/mL, *p* = 0.029) (Figure 2). ders. The comparison of serum suPAR levels among groups of asthmatics is presented in Table 1. suPAR levels were significantly higher in asthmatics with severe uncontrolled asthma not receiving biologics than in patients with moderate uncontrolled asthma (3.3 ± 0.7 vs. 2.1 ± 0.4 ng/mL, *p* = 0.023) (Figure 1). Moreover, suPAR levels were significantly

sodium, potassium, urea, creatinine, AST, ALT, γGT, and ALP were detected among gen-

No correlations were found between suPAR levels and age, BMI, T2 biomarkers, white blood cell count, CRP, electrolytes, parameters of the kidney and liver function, or spirometric parameters (Supplementary Table S1). The correlation analysis between suPAR levels and the most important parameters is shown in Figure 3. lower in asthmatics with moderate uncontrolled asthma than in patients with severe uncontrolled asthma without (2.1 ± 0.4 vs. 3.3 ± 0.7 ng/mL, *p* = 0.023) or with biologics (2.1 ± 0.4 vs. 3.6 ± 0.8 ng/mL, *p* = 0.029) (Figure 2).

Note: \* *p* = 0.023

**Figure 1.** Comparison of serum soluble urokinase plasminogen activator receptor (suPAR) levels between groups of asthmatics. **Figure 1.** Comparison of serum soluble urokinase plasminogen activator receptor (suPAR) levels between groups of asthmatics.

Note: \* *p* < 0.05

**Figure 2.** Comparison of serum ssoluble urokinase plasminogen activator receptor (suPAR) levels between moderate uncontrolled and severe uncontrolled asthmatics. **Figure 2.** Comparison of serum ssoluble urokinase plasminogen activator receptor (suPAR) levels between moderate uncontrolled and severe uncontrolled asthmatics. white blood cell count, CRP, electrolytes, parameters of the kidney and liver function, or spirometric parameters (Supplementary Table S1). The correlation analysis between su-PAR levels and the most important parameters is shown in Figure 3.

between moderate uncontrolled and severe uncontrolled asthmatics.

**Figure 2.** Comparison of serum ssoluble urokinase plasminogen activator receptor (suPAR) levels

No correlations were found between suPAR levels and age, BMI, T2 biomarkers,

*J. Pers. Med.* **2022**, *12*, x FOR PEER REVIEW 5 of 9

**Figure 3.** Correlation analysis between suPAR levels and age, body mass index (BMI), T2 biomarkers, and forced expiratory volume in the first second (FEV1)/ forced vital capacity (FVC). Note: No correlation was found between suPAR levels and fractional exhaled nitric oxide (FeNO) (r = −0.014, *p* = 0.308), eosinophils % (r = −0.037, *p* = 0.767), eosinophil count (r = −0.016, *p* = 0.895), age (r = 0.14, *p* = 0.238), BMI (r = 0.079, *p* = 0.646)), and FeV1/FVC (r = 0.020, *p* = 0.870). Abbreviations: BMI, body mass index; FeNO, fractional exhaled nitric oxide; FeV1/FVC, forced expiratory volume in the first second/forced vital capacity.

#### **4. Discussion**

This study found that suPAR levels were significantly higher in asthmatics with severe uncontrolled asthma with or without biologics than in patients with moderate uncontrolled asthma. No correlations were found between suPAR levels and age, BMI, T2 biomarkers, white blood cell count, CRP, electrolytes, kidney and liver function parameters, CRP, or spirometric parameters.

Previous studies have shown that suPAR is associated with disease progression and severity in multiple diseases. Thus far, few studies have explored suPAR's role in asthma outcomes [1]. More specifically, elevated suPAR levels have been associated with hospital all-cause readmission and all-cause mortality in hospitalized patients with a diagnosis of asthma made as soon as they were acutely admitted to the emergency department [8]. Another study found that suPAR concentrations were increased in a small cohort of asthmatics with poor disease control compared to patients with well-controlled asthma [1].

In this study, we found no correlation between age and suPAR levels. Nevertheless, there is evidence that age is a non-modifiable risk factor that correlates with an increase in su-PAR levels [14]. A previous study in a population of 182 generally healthy individuals aged 74–89 years found that those aged 24–66 years had higher suPAR levels than younger controls: 3.79 ng/mL (95% CI 3.64–3.96 ng/mL) vs. 3.16 ng/mL (95% CI 2.86–3.45 ng/mL) [14]. These levels increased further with advancing age and were similar in women and men. Aging is associated with systematic cardiac and vascular structure alterations due to immunological responses and natural hormonal changes, resulting in a gradual decline in organ function [14]. However, in our study, we excluded asthmatics with comorbidities associated with low-grade chronic inflammation processes such as diabetes, heart failure, and malignant and inflammatory systemic diseases; this could explain the fact that we did not find any association between age and suPAR levels.

Although obesity is considered a low-grade inflammatory disease, or parainflammation, in this study, we found no association between suPAR levels and BMI. Limited data investigate suPAR as an inflammatory biomarker in obesity. More specifically, Kosecik et al. reported that suPAR has no predictive value for future atherosclerosis in obese children after investigating 136 participants with a median age of 12.05 years [15].

We found no association between suPAR levels and T2-high biomarkers. A previous study documented that in patients acutely admitted with asthma, elevated suPAR concentrations together with blood eosinophil count < 150 cells/µL at the time of hospital admission were associated with both 365-day all-cause readmission and mortality, implying that in asthma, the uPAR pathway associates with non-T2 asthma but is implicated in neutrophils and T1/T17 T-cells that are thought to be part of the pathogenesis of the non-T2 asthma endotypes [14,16,17].

Neutrophils are a primary source of circulating suPAR [18]. Studies report the usefulness of suPAR in predicting severe outcomes in critical illness related to inflammatory and infectious diseases [18], along with CRP and leukocytes. In this study, we found no correlation between suPAR and other inflammatory markers, such as white blood cell count or CRP, given that we excluded patients with significant comorbidities. In the same context, no correlation was detected between suPAR and kidney parameters and liver function parameters. However, there is evidence that suPAR plasma levels were significantly higher in patients with chronic kidney disease (7.9 ± 3.82 ng/mL) than in controls (1.76 ± 0.77 ng/mL, *p* < 0.001) and correlated with disease severity [19]. Similar to its prognostic properties in patients with sepsis, serum suPAR concentrations might serve as an interesting biomarker in cirrhosis and acute liver failure [19].

FEV1 levels are not the only factor taken into account to classify disease severity [16]; they have long been known to be one of the major predictors of mortality among individuals with asthma [16]. However, lung function deficits with magnitudes insufficient to cause clinically manifest functional impairments found in mild asthma are also related to molecular pathways that increase susceptibility to the pulmonary effects of exposures. Furthermore, small airway disease (SAD) is highly prevalent in asthma, even in patients

with milder disease. Structural alterations at the peribronchiolar level contribute to the pathogenesis of functional abnormalities observed in patients with asthma [16]. Remodeling can affect small airway wall stiffness, thereby changing their distensibility. Given the clinical impact of SAD, its presence should not be underestimated or overlooked as part of the daily management of patients with asthma. SAD is likely to be directly or indirectly captured by combinations of physiological tests, such as spirometry. Notably, suPAR levels have been previously linked to impaired lung function and airway resistance [1]. This study found no correlation between suPAR levels and airway obstruction. Further research is needed to evaluate any potential correlation between suPAR levels and SAD using more detailed techniques.

In asthma, a panel of several cytokines, chemokines, and granule proteins induce airway inflammation and hyperresponsiveness through enhancing innate and type 2 (T2) or non-T2 immune responses [1,8,16]. Although disease severity-related airway inflammation is found in asthma, new evidence has documented persistent chronic airway inflammation and remodeling in mild asthma, except for those with severe asthma, as defined by the treatment step [8]. Neutrophilic asthma is the lesser-known asthma phenotype and is characterized by severe refractory disease. Airway neutrophilia is associated with asthma severity, acute asthma exacerbation, and airflow limitation. However, neutrophils can also be detected in the airways of mild asthmatics [8,16]. Interestingly, studies suggest that the inflammation reflected in circulating suPAR concentrations in part stems from neutrophil activity [9], commonly considered to be non-T2 inflammation [16]. Accordingly, this study found no correlation between suPAR concentrations and T2 biomarkers such as eosinophils and FeNO.

Our study's findings should be interpreted within the context of its limitations. As such, when considering absolute numbers, our study's population is small, in a single center with patients from the same population in terms of geo-ethnicity, limiting our findings' generalizability. Larger multi-center and multi-nation studies are needed to confirm our results. However, other factors could not have confounded our findings, given that we carefully excluded patients with comorbidities associated with high suPAR levels.

#### **5. Conclusions**

suPAR levels were higher in asthmatics with severe disease receiving or not receiving biologics than in those with moderate uncontrolled asthma. suPAR's high stability in plasma samples and its noninvasiveness make it an ideal candidate for the management of asthma and the prediction of worse outcomes. The findings of this study suggested a prognostic value of suPAR that would translate into clinical practice in asthma patients and might predict step-up treatment benefits across the spectrum of asthma severity. Furthermore, the information can be used to develop targeted interventions aimed at the risks of so-called mild asthma. An important practical implication is that suPAR might be a useful addition to existing stratification algorithms for identifying patients that particularly benefit from step-up treatment. This study also indicates that suPAR levels might be effective for clinical use associated with specific clinical features, inflammatory phenotypes of asthma, or impaired lung function.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jpm12111776/s1.

**Author Contributions:** Conceptualization, O.S.K.; methodology, O.S.K. and I.P.; validation, O.S.K.; I.P.; formal analysis, O.S.K.; investigation, G.M., K.T., K.M., P.K.; data curation, O.S.K.; writing—original draft preparation, O.S.K.; writing—review and editing, O.S.K.; supervision, O.S.K., K.I.G., Z.D.; project administration, O.S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is supported by a Grant from the Hellenic Thoracic Society. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. (Grant Number: 17/2020).

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Thessaly (No. 2800- 01/11/2020; approved on 1 November 2020).

**Informed Consent Statement:** Patient consent was waived due to the retrospective nature of this study, and the analysis used anonymous clinical data.

**Data Availability Statement:** The data that support the findings of this study are available on request from the corresponding author, O.S.K.

**Conflicts of Interest:** The authors declare no conflict of interest.

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