# ENVIRONMENT AND HEALTH

EDITED BY : Mario Sprovieri, Ethel Eljarrat and Fabrizio Bianchi PUBLISHED IN : Frontiers in Public Health, Frontiers in Earth Science and Frontiers in Environmental Science

#### Frontiers eBook Copyright Statement

The copyright in the text of individual articles in this eBook is the property of their respective authors or their respective institutions or funders. The copyright in graphics and images within each article may be subject to copyright of other parties. In both cases this is subject to a license granted to Frontiers. The compilation of articles constituting this eBook is the property of Frontiers.

Each article within this eBook, and the eBook itself, are published under the most recent version of the Creative Commons CC-BY licence. The version current at the date of publication of this eBook is CC-BY 4.0. If the CC-BY licence is updated, the licence granted by Frontiers is automatically updated to the new version.

When exercising any right under the CC-BY licence, Frontiers must be attributed as the original publisher of the article or eBook, as applicable.

Authors have the responsibility of ensuring that any graphics or other materials which are the property of others may be included in the CC-BY licence, but this should be checked before relying on the CC-BY licence to reproduce those materials. Any copyright notices relating to those materials must be complied with.

Copyright and source acknowledgement notices may not be removed and must be displayed in any copy, derivative work or partial copy which includes the elements in question.

All copyright, and all rights therein, are protected by national and international copyright laws. The above represents a summary only. For further information please read Frontiers' Conditions for Website Use and Copyright Statement, and the applicable CC-BY licence.

ISSN 1664-8714 ISBN 978-2-88966-207-4 DOI 10.3389/978-2-88966-207-4

### About Frontiers

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

### Frontiers Journal Series

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

### Dedication to Quality

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

### What are Frontiers Research Topics?

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office<EMAIL_ADDRESS>
# ENVIRONMENT AND HEALTH

Topic Editors: Mario Sprovieri, Italian National Research Council, Italy Ethel Eljarrat, Institute of Environmental Assessment and Water Research (CSIC), Spain Fabrizio Bianchi, Italian National Research Council, Italy

Citation: Sprovieri, M., Eljarrat, E., Bianchi, F., eds. (2020). Environment and Health. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88966-207-4

# Table of Contents

Maria Catrambone, Paolo Cristofanelli, Stefano Dalla Torre, Giulio Esposito, Marco Giusto, Silvia Mosca, Salvatore Pareti, Elena Rantica, Tiziana Sargolini, Giorgio Tranchida and Paolo Bonasoni

*91 Indoor Particulate Matter From Smoker Homes Induces Bacterial Growth, Biofilm Formation, and Impairs Airway Antimicrobial Activity. A Pilot Study* Emma M. Stapleton, Robert Manges, Gavin Parker, Elizabeth A. Stone, Thomas M. Peters, Robert J. Blount, Julio Noriega, Xiaopeng Li, Joseph Zabner, Philip M. Polgreen, Octav Chipara, Ted Herman and Alejandro P. Comellas

Antonella Ausili, Luisa Bergamin and Elena Romano

Calogero Di Bella, Anna Traina, Cristina Giosuè, Davide Carpintieri, Gianluigi Maria Lo Dico, Antonio Bellante, Marianna Del Core, Francesca Falco, Serena Gherardi, Maria Michela Uccello and Vincenzo Ferrantelli

*175 Persistent and Emerging Organic Pollutants in the Marine Coastal Environment of the Gulf of Milazzo (Southern Italy): Human Health Risk Assessment*

Fabio D'Agostino, Antonio Bellante, Enza Quinci, Serena Gherardi, Francesco Placenti, Nadia Sabatino, Gaspare Buffa, Giuseppe Avellone, Vita Di Stefano and Marianna Del Core

*188 Linking Bioeconomy to Redevelopment in Contaminated Sites: Potentials and Enabling Factors*

Fedra Francocci, Fabio Trincardi, Andrea Barbanti, Massimo Zacchini and Mario Sprovieri

# Editorial: Environment and Health

Mario Sprovieri 1 \*, Ethel Eljarrat 2 and Fabrizio Bianchi 3,4

1 Institute of Anthropic Impacts and Sustainability in the Marine Environment (IAS-CNR), Capo Granitola, Italy, 2 Institute of Environmental Assessment & Water Research (IDAEA-CSIC), Barcelona, Spain, 3 Institute of Clinical Physiology (IFC-CNR), Pisa, Italy, 4 Institute for Biomedical Research and Innovation (IRIB), Palermo, Italy

Keywords: environment human health, air quality, marine environment, circular bioeconomy, remediation and restoration

Editorial on the Research Topic

Environment and Health

### INTRODUCTION

Ten thousand years of protracted and increasing interaction between humans and their environment has influenced, beside the quality of ecosystems, also our quality of life, healthy life span, and health inequalities. In 2016 WHO reported that globally some 12.6 million deaths each year can be ascribed to unhealthy environments. Moreover, 24% of global deaths (and 28% of deaths among children under five) come from modifiable environmental factors (Prüss-Ustün et al., 2016). Human epidemiological studies and experiments in laboratory animals proved that exposure to some pollutants can increase susceptibility to diseases such as diabetes, heart disease, and reproductive cancers. Exposures to toxic agents can also cause immunosuppression, which increases vulnerability to infections, such as COVID-19 (Birnbaum and Heindel, 2020). Environmental factors which represent specific threats for humans and ecosystems are directly associated to exposure to hazardous substances in air, water, soils, and food. Marine sediments and seawater represent an additional major source of contaminants to the environment through multiple pathways. Risks and consequences are also amplified by climate changes and linear and non-linear combinations are reflected in multi-hazard effects. Consequences of living and working in a poor-quality environment could be further magnified in groups of people with poorer health and socio-economic status. In this view, the strategic framework of Agenda 2030 launched by UN refers to the Sustainable Development Goals as critical sectors where all Countries are urged to promote actions to protect the planet (https://www.who.int/topics/millennium\_development\_goals/en/). This comprehensive vision recognizes that building economic growth requires address a range of social needs including education, social protection and job opportunities, while tackling climate change and environmental defense. The highly integrated 17 Sustainable Development Goals represent a solid framework that ensuring healthy lives and promoting well-being at all ages is essential to sustainable development. Major progress was made in improving the health of millions of people and relevant efforts were also oriented in increasing life expectancy and reducing some of the common negative impacts. At the same time, worldwide consumption and production depend on the use of the natural environment and resources in a way that continues to have destructive impacts on the planet. Economic and social progress over the last century has been accompanied by environmental degradation that is endangering the ecological systems on which the human future depends. Thus, the development of multidisciplinary approaches for understanding mechanisms and dynamics of interference between environment and health is a crucial commitment. The holistic "One Health" approach (e.g., Gibbs and Paul, 2014; CE29.6.2017-COM, 2017 COM 339 Final, 2017) combining efforts of WHO, Food and Agriculture Organization, and World Organization for Animal Health,

Edited and reviewed by: Martyn Tranter, University of Bristol, United Kingdom

> \*Correspondence: Mario Sprovieri<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Geochemistry, a section of the journal Frontiers in Earth Science

Received: 25 August 2020 Accepted: 07 September 2020 Published: 09 October 2020

#### Citation:

Sprovieri M, Eljarrat E and Bianchi F (2020) Editorial: Environment and Health. Front. Earth Sci. 8:598611. doi: 10.3389/feart.2020.598611 promotes multi-sectorial responses to food safety hazards, risks from zoonoses, and other public health threats. Thus, the humananimal-ecosystem-human interfaces represent primary fields of investigation for valuable improvements of the well-being of human population, ecosystem and environment.

This Research Topic comprises 14 studies including original research articles, method developments, reviews, and perspectives covering several aspects of the complex interaction between environment and health, with critical reviews on key environmental sectors as outdoor and indoor air quality, food, and marine environments. Also, specific proposals of modern approaches to monitor environment and health outcomes as well as restoration and recovery of polluted environments represent a crucial part of this topic. The collection contributes to create and implement effective conceptual links and "bridges" between distinct scientific communities, working in a multidisciplinary way. Most of the articles concern air and sea, as well as some insights dedicated to modern strategies to restore and recover polluted environment, also considering the impacts on human health and the economy.

### AIR QUALITY

Poor air quality leads to premature death, cancer, and long-term damage to respiratory and cardiovascular systems. WHO estimates that environmental air pollution caused some 4.2 million deaths in 2016, while household air pollution from cooking with polluting fuels and technologies caused an estimated 3.8 million deaths in the same period (https://www. who.int/health-topics/air-pollution#tabtab\_1). Progress has been made to reduce unhealthy air emissions, an important step in creating a healthier environment. In particular, the Declaration of the Sixth Ministerial Conference on Environment and Health (held in Ostrava, Czech Republic, on June 15, 2017) includes a commitment to "prevent and eliminate adverse effects on the environment and health, costs and inequalities relating to waste management and contaminated sites, progressing toward the elimination of the disposal and trafficking of uncontrolled and illegal waste, and the proper management of waste and contaminated sites in the context of the transition toward a circular economy" (Sixth Ministerial Conference on Environment and Health. Available at: http:// www.euro.who.int/en/media-centre/events/events/2017/06/ sixth-ministerial-conference-on-environment-and-health). In this context, Manisalidis et al. (2020) documented, through a comprehensive knowledge-based analysis, modes and dynamics of air pollution and related impact on human health (at different systems level) and suggests specific science to policy actions as valuable trajectories to sustainable solutions. Perrino et al. (2020) measured continuous trace gases and erosol (including NOx, SO2, O3, NMHC) organic and inorganic pollutants on particulate matter (PM10 and PM2.5) over the period 2016–2018 from two highly polluted sites in southern Italy and offer original insights and understanding on the role of anthropogenic emissions and particularly on mechanisms of ozone formation, also considering the WHO limits. Kim et al. (2020) studied risk factors correlated to outdoor air pollutant exposure (PM10, PM2.5, and NO2), specifically in relation to cognitive impairment in elderly and gender-differentiated population of South Korea. The preliminary results suggest higher adverse effects of outdoor air pollution on cognitive function in women, indicating specific actions for prevention and intervention.

Stapleton et al. (2020) reported on a pilot study in 21 homes selected from the NIH funded cohort residing in Iowa of current and former smokers, with and without a history of respiratory exacerbations. The aim of the research was at assessing whether indoor air particulate collected from their homes would affect in vitro bacterial growth, biofilm formation, and primary human airway surface liquid antimicrobial activity, identifying mechanisms in the development of respiratory tract infections, including bacterial growth, biofilm formation, and innate immunity. Herting et al. (2020) assessed a systematic review on the available literature on the evidence from Magnetic Resonance Imaging studies on how early-life exposure responds to outdoor air pollution on neuro-development. Correlations between pollutants and physiological brain features suggested that outdoor air pollution may significantly affect structure and function of brain. Further air pollutionneuroimaging studies are urgently needed in a developmental neuroscience perspective. Viegas et al. (2020) presented a comprehensive study on the effects of mineral sulfurous thermal waters on lung epithelial-immune crosstalk through the action of its main component, H2S as valuable inhalational treatment of respiratory diseases.

### Marine Environment and Human Health

The marine environment is essential for human health through the provision and quality of air, food and water and offering health-enhancing economic and recreational opportunities. However, human activities such as transport, industrial processes, agricultural and waste management exerts an increasing pressure on marine environment. Many knowledge gaps still significantly limit planning of policies orienteered to a sustainable use of marine resources and environmental and human health protection. The European Marine Board (2013) reported a global estimate of 250 million cases of gastroenteritis from bathing in contaminated water, and 50,000–100,000 annual deaths caused by infectious hepatitis. The global burden of human disease caused by sewage pollution of coastal waters has been estimated at four million casualties, annually. The knowledge about the behavior and ecotoxicity of pollutants (including those of emerging concern) in the marine environment is particularly relevant given the complex interactions among different matrices (sediments, seawater, atmosphere, and biota), and represents a field of cutting-edge multidisciplinary science. In this context, Ausili et al. (2020) presented a comprehensive view on the current status of monitoring and recovery actions of highly industrialized coastal-marine areas in Italy, and provide quantitative indicators to inform policies for restoring huge territory affected by pollution. D'Agostino et al. (2020) reported on the environmental status of contamination by persistent and emerging contaminants (polycyclic aromatic hydrocarbons, Pesticides, polybrominated diphenyl ethers, etc.) from a Sprovieri et al. Editorial: Environment and Health

highly contaminated site in southern Italy, and offer new approaches to estimate cancer and non-cancer human health risk due to dermal absorption from contaminated seawater and/ or ingestion of contaminated fish. Mekni et al. (2020) examined a mixture of legacy (polybrominated diphenyl ethers) and emerging contaminants, such as halogenated (norbornenes (HNs)) and organophosphate (OPFRs) flame retardants, in sediments and samples of eel (Anguilla anguilla) from the Tunisian Bizerte Lagoon. Chemical behavior at the environmental interfaces has been explored and health risk associated to the consumption of eel has been estimated, offering original methodological approaches to evaluate impacts by new classes of pollutants in the marine environment. Simmons et al. (2020) reported on a beadbased salivary IgG antibody multiplex immunoassay to determine and quantify infections from environmental exposures to six waterborne pathogens. Results refer to a wide spectrum of 2,091 study participants at Boquerón Beach, Puerto Rico during the summer of 2009 and show how simultaneous infections could affect human health, in synergistic and/or antagonistic interactions. Combined to water quality studies, environmental microbial pathogenesis provides valuable risk assessment tools in estimating exposure potential and facilitating the development of disease surveillance and screening tools.

### Strategies for Solutions

The scientific community is urged to provide valuable knowledge about the dynamics of pollutants in the environment but also to offer solutions at short to mid-term to improve environmental quality and ecosystem and human health. In this view, Drago et al. (2020) reported preliminary results from a new birth cohort in highly contaminates sites in southern Italy. The approach offered an unprecedented opportunity to monitor effects of integrated environmental effects on the health of mother-son

### REFERENCES

couples in the short- to long-term period. Di Bella et al. (2020) presented original results about a suite of organic and inorganic contaminants detected in food (terrestrial and seafood) from highly contaminated sites in southern Italy offering original approaches to assessment of associated health risk for different age profiles. Moxley et al. (2020) presented detailed results on the biodegradation efficiency of halogenated compounds in contaminated soils using groups of meso- to thermophilic microorganisms under the extreme conditions of arid and semi-arid areas. Francocci et al. (2020) presented a comprehensive view and original applications of the circular bioeconomy applied to contaminated sites as valuable holistic solution for restoration/recovery of large polluted sites.

### CONCLUSIONS

This Research Topic should inspire future science trajectories focused on multi- and interdisciplinary investigations to meet environment restoration and healthier societies. The complexity and significant heterogeneity of the contributions reinforce the urgency to broaden and integrate science approaches and visions on the relationship between environment and health. An issue of increasing priority and urgency.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### ACKNOWLEDGMENTS

We thank all authors, reviewers, and editors that have contributed to this Research Topic.

activity. A pilot study. Front. Public Health 7. doi:10.3389/fpubh.2019. 00418

Viegas, J., Esteves, A. F., Cardoso, E. M., Arosa, F. A., Vitale, M., and Taborda-Barata, L. (2020). Biological effects of thermal water-associated hydrogen sulfide on human airways and associated immune cells: implications for respiratory diseases. Front. Public Health 7. doi:10.3389/fpubh.2019.00128

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Sprovieri, Eljarrat and Bianchi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Influence of Abiotic Factors Temperature and Water Content on Bacterial 2-Chlorophenol Biodegradation in Soils

#### Ellen Moxley 1,2, Elena Puerta-Fernández 2 , Enrique J. Gómez 2 and Juan M. Gonzalez 2 \*

1 Villanova University, Villanova, PA, United States, 2 Department of Biogeochemistry, Plant and Microbial Ecology, Instituto de Recursos Naturales y Agrobiologia de Sevilla, IRNAS-CSIC, Seville, Spain

Halogenated compounds are environmental pollutants toxic to humans and wildlife. Certain microorganisms degrade these halogenated compounds. However, little is

known about the potential of microorganisms in bioremediation under extreme conditions, specifically in arid and semi-arid soils frequently exposed to high temperatures and desiccation periods. Arid and semi-arid environments and deserts make up vast areas of Earth's landmass. To investigate the degradation of 2-chlorophenol (2-CP) in soils as a function of temperature and water availability, three bacterial species were tested, two soil mesophiles of the genus Rhodococcus, R. opacus and R. erythropolis, and a soil thermophilic isolate, Parageobacillus thermoglucosidasius. Degradation trials in soil samples with these species were performed over a range of water activity from 1 to 0.4. At their optimum growth temperature, R. opacus showed maximum 2-CP degradation at water activity 0.9 sharply decreasing when lowering water activity. Nevertheless, the Parageobacillus isolate (optimum growth temperature 60◦C) showed maximum 2-CP degradation rates at water activity 0.5 which represented highly desiccating conditions. Parageobacillus degradation of 2-CP was very low at water activity above 0.9. Thus, biodegradation of 2-CP in soils is possible even under arid conditions although different microbial species might be involved in this task depending on the interactions of abiotic factors and the diversity of microbial communities in soils. These results contribute to understand the potential biodegradation of specific halogenated compounds in the environment which is of great relevance to comprehend the fate of halogenated pollutants (i.e., 2-CP) in deserts, arid and semi-arid soils.

Keywords: 2-chlorophenol, temperature, water availability, desiccation, soil, biodegradation Rhodococcus, Parageobacillus, bioremediation

### INTRODUCTION

Halogenated compounds, such as chlorophenols, represent important environmental pollutants. Chlorophenols are released into the environment through anthropogenic activities, for instance by their use in pesticides, herbicides, their presence in industrial wastes (Arora and Bae, 2014) and also as a result of organic matter combustion and biological chlorination (Ahling and Lindskog, 1982). Chlorophenols can adhere onto particulate organic matter persisting in the environments for

#### Edited by:

Ethel Eljarrat, Institute of Environmental Assessment and Water Research (CSIC), Spain

#### Reviewed by:

Gulnihal Ozbay, Delaware State University, United States Jean Challacombe, Colorado State University, United States

> \*Correspondence: Juan M. Gonzalez<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Environmental Health, a section of the journal Frontiers in Environmental Science

> Received: 17 January 2019 Accepted: 14 March 2019 Published: 04 April 2019

#### Citation:

Moxley E, Puerta-Fernández E, Gómez EJ and Gonzalez JM (2019) Influence of Abiotic Factors Temperature and Water Content on Bacterial 2-Chlorophenol Biodegradation in Soils. Front. Environ. Sci. 7:41. doi: 10.3389/fenvs.2019.00041

**9**

extended periods of time and leading to accumulation in the organisms through different trophic levels. Understanding the fate of chlorophenols in nature is complex and it is certainly the result of an interplay of biotic and abiotic processes.

The biological degradation of chlorophenols has been studied (Arora and Bae, 2014) and several microorganisms have been reported to be able to mineralize chlorophenols (Arora and Bae, 2014). The extent to what these microorganisms could perform chlorophenol degradation in the natural environment remains to be fully understood. In addition, the influence and effect of multiple abiotic factors needs to be added to our comprehension of chlorophenol degradation to be able to provide a knowledgeable management of bioremediation procedures and models on the environmental fate of chlorophenol.

Soils are highly heterogeneous environments (Or et al., 2007) holding a huge microbial diversity (Curtis et al., 2002). As a consequence, following the fate of chlorophenols in these environments is a complex task. Environmental factors, such as temperature and water content, are important contributors to soil heterogeneity and changes in microbial activity (Manzoni et al., 2012) which must directly influence soil biodegradation of chlorophenols. Nevertheless, the influence of these factors and their interplay with specific chlorophenoldegrading microorganisms remains to be understood.

Meteorological factors can influence the functioning and mechanisms occurring at upper soil layers. For instance, high temperature and sun exposure can result in elevated temperature measurements at upper soil layers (Portillo et al., 2012), which implies the activation of soil thermophilic bacteria (Portillo et al., 2012), and, consequently, cause a switch on the functionally dominant bacteria and metabolic processes (Portillo et al., 2012; Santana and Gonzalez, 2015). This scenario can have important consequences both at local and global scales (Santana and Gonzalez, 2015). In addition, high temperature events imply increased evaporation leading to soils with low water availability, desiccation and aridity. Temperature and water content are intimately related at upper soil layers and could be implicated in important consequences on the behavior of soil microbial communities (Manzoni et al., 2012; Biederman et al., 2016), including their potential for biodehalogenation. There is a current gap in our knowledge on the influence of these factors (temperature and water availability) on the microbial potential to decompose chlorophenols.

The extension of landmass assigned to deserts, arid and semiarid ecosystems represents a major area of terrestrial Earth surface located mostly at medium and low latitudes (Portillo et al., 2012; Santana and Gonzalez, 2015). Many of the aridclassified sites are potential targets for halogenated compound disposal in the field because these lands frequently belong to under-developed countries or present relatively low inhabiting human population. In this scenario, understanding the fate of chlorophenols in dry terrestrial environments is an important concern to envision if these pollutants are to be decomposed or will long persist in these environments.

This study will focus on 2-chlorophenol (2-CP) as a case study of biodehalogenation to better understand the fate of this pollutant in soils. We directed special attention to the effect of temperature and desiccating conditions on the fate of 2-CP in the environment. The aim of this study is to assess the influence of high temperature and low water content on the differential removal of 2-CP by distinct soil bacteria as a first approach to understand the potential bioremediation of halogenated pollutants, above all, in arid and dry soils.

### MATERIALS AND METHODS

### Bacterial Strains and Soil Samples

Two mesophilic (optimum growth temperature 28◦C), 2- CP-degrading strains of the genus Rhodococcus, R. opacus IG (DSM 43205) (Klatte et al., 1994) and R. erythropolis DSM 43066 (Goodfellow and Anderson, 1977), were used in this study as soil isolates previously evaluated for their ability to degrade 2-CP (Bondar et al., 1999; Goswami et al., 2002; Arora and Bae, 2014). These strains were grown in medium 535 (Klatte et al., 1994) as recommended by the DSMZ (German Collection of Microorganisms and Cell Cultures) which composition (per liter) included: pancreatic digest of casein (17.0 g), enzymatic digest of soy bean (3.0 g), sodium chloride (5.0 g), dipotasium hydrogen phosphate (2.5 g), and glucose (2.5 g). Medium 535 was adjusted to pH 7.3. In addition, a soil thermophilic isolate (optimum growth temperature 60◦C), P. thermoglucosidasius strain 23.6 (previous Geobacillus) (Aliyu et al., 2016) was a representative of the ubiquitous thermophilic bacterial community detected in soils (Marchant et al., 2002; Portillo et al., 2012) and grown in Nutrient Broth which contains (per liter): beef extract (5.0 g), peptone (10.0 g), and sodium chloride (5.0 g). Nutrient Broth was adjusted to pH 7.2 at 60◦C. Strain 23.6 was confirmed to decompose 2-CP in liquid medium.

Soil samples were collected at Coria del Rio (Sevilla, Spain) at the experimental agricultural area "La Hampa" (IRNAS-CSIC) which soil characteristics have been previously reported (Cardoso de Barros, 1996).

### Biodegradation of 2-Chlorophenol

Most studies have performed estimates of dehalogenation in aqueous solution. Unlike them, herein, we present a more realistic analysis by quantifying decomposition of a halogenated compound (i.e., 2-CP) in soils considering its high physical and chemical heterogeneity. The procedure allows to evaluate decomposition of 2-CP (and similarly other halogenated pollutants) at different water availabilities (i.e., water activity). To evaluate the decomposition of 2-CP over time by the studied strains under realistic soil conditions, 400 mg of autoclaved soil (3 consecutive days each at 121◦C for 20 min) were supplemented with 2-CP (50 µl, 0.5 g/l) and 70 µl of a culture at its exponential phase of growth. Controls lack the addition of cells which were replaced by the same volume of sterile distilled water. The soil plus cell mixtures were desiccated in a vacuum concentrator (Micro-Cenvac, N-Biotek, Gyeonggi-do, South Korea) to obtain the desired water activity (aw). Once the desired aw was obtained, the samples (in triplicates) were placed in an incubator to the

required growth temperature (either 20 or 60◦C for mesophiles or thermophiles, respectively). Over time, samples were collected at 0, 4, 8, 24, and 72 h incubation to determine the concentration of 2-CP in the soil mixtures.

Water activity represents the water available for microorganisms and it is defined as the partial vapor pressure of water in a sample divided by the partial vapor pressure of pure water, and it ranges from 0 (no water) to 1 (water). The aw was determined using a water activity probe (Rotronic AG, Bassersdorf, Switzerland). The soil sample to be determined was placed in the temperature controlled probe chamber to perform the measurement following the manufacturers' recommendations. As a reference, the minimum aw that allows the growth of a microorganism (the fungus Xeromyces bisporus) is >0.63 (Stevenson et al., 2015).

Quantification of 2-CP was carried out by HPLC (Agilent Infinity 1260; Agilent, Santa Clara, California, USA) using a mobile phase consisting on Acetonitrile:Water (70:30), and an injection volume of 10 µl through a column Agilent Zorbax Eclipse Plus C18 (Agilent, USA) at 35◦C. Soil aliquots collected over time were supplemented with 1 ml acetonitrile to dissolve 2-CP, sonicated at 40◦C for 15 min and vortex at room temperature for additional 15 min. The suspension was centrifuged at 8,000 × g for 4 min. The supernatant was collected and filtered through a 0.2 µm-pore-diameter filter. The extracted solution was injected in the described HPLC system and 2-CP quantified by peak integration after absorbance measurement at 273 nm (Goswami et al., 2002). 2-CP quantifications over time were used to estimate the decomposition rate (per hour) through the linear portion of the decay curve (**Figure 1**). These decomposition rates were estimated by linear regression (Sokal and Rohlf, 1995). All experiments were carried out in independent triplicates and the averages and standard deviations are presented.

### RESULTS AND DISCUSSION

Understanding the functioning of soil ecosystems under a variety of environmental conditions is a requirement for a proper management of soils. Soils can be used as a source for economical benefit and food products as well as a potential site for the biodecomposition of human activity pollutants. Soils, and specifically, semi-arid and arid soils, are potential targets for dumping halogenated pollutants. Their environmental risks are high and it is of most importance to understand when and how the biodecomposition of 2-CP (as well as other halogenated pollutants) occurs in these soils.

Environmental conditions, such as high temperature and dry periods, affect soil upper layers. Besides, these upper layers are the most influenced by pollutants. However, the range of environmental conditions allowing bacteria to biodegrade halogenated compounds remains to be studied. Herein, we approach the biodegradation of 2-CP in a soil by different soil bacteria under a variety of temperature and water availability conditions commonly observed in natural soils.

The analysis of the capability of Rhodococcus species to decompose 2-CP in natural soils revealed that these soil mesophiles are only able to carry out efficient biodegradation of 2-CP under soggy and temperate conditions (**Figure 2**). Nevertheless, soil upper layers frequently reach high temperatures (e.g., up to 75◦C during summer in Southern Spain and over 90◦C at desserts) (McCalley and Sparks, 2009; Portillo et al., 2012) and consequently, their water availability is highly reduced, reaching highly desiccating values. Under these frequently encountered soil conditions, it is unknown if bacteria can carry out the process of decomposition of halogenated pollutants in soils and which bacteria and under which conditions this could be processed. Common 2-CP decomposing soil bacteria, such as R. opacus and R. erythropolis,

FIGURE 2 | Biodegradation of 2-CP in soil as a function of water activity for three bacterial species: a thermophilic Parageobacillus thermoglucosidasius (black continuous line and squares), and two mesophiles, Rhodococcus opacus (black discontinuous line and triangles) and R. erythropolis (gray discontinuous line and triangles). Note that the optimum 2-CP decomposition rate for the thermophilic species, P. thermoglucosidasius, was observed at water activity 0.5 (corresponding to a quite dry soil). For Rhodococcus, the optimum 2-CP decomposition rate occurred under soggy conditions, at water activity >0.9. Error bars represent one standard deviation.

are unable to carry out efficient decomposition of 2-CP in dry soils and at high soil temperatures (**Figure 2**).

Nevertheless, our results have shown that common soil thermophilic bacteria (i.e., Parageobacillus) (Portillo et al., 2012; Santana and Gonzalez, 2015; Aliyu et al., 2016) are capable of efficiently biodecompose 2-CP under high temperature and dry conditions commonly observed at upper soil layers (**Figure 2**). This suggests that thermophilic bacteria could replace mesophiles during hot soil periods. In fact, soils can get frequently hot at medium and low latitudes which is coincident with those land areas on Earth most exposed to high temperatures (Gonzalez et al., 2015; Santana and Gonzalez, 2015). These results confirm that under arid conditions, some soil bacteria can continue the biodegradation process of halogenated pollutants (i.e., 2-CP) and so these polluted soils can be bioremediated even under extreme temperature and water scarcity conditions.

Previous reports have indicated that thermophilic bacteria are ubiquous in soils (Marchant et al., 2002; Portillo et al.,

### REFERENCES

2012). This presence of thermophilic bacteria in soils has explained the observation that maximum enzymatic hydrolytic activities in soils always occur at high temperatures (Gonzalez et al., 2015) and this has been related to soils with reduced organic matter content (Santana and Gonzalez, 2015). Those results represented previous evidence on the great potential of soil thermophiles in soils functioning and, definitively, in soil health (Marchant et al., 2002; Portillo et al., 2012; Santana and Gonzalez, 2015; Wong et al., 2015), even on their potential for dechlorination (Larsen et al., 1991) and the decomposition of recalcitrant hydrocarbons (Wong et al., 2015) in soils. Consequently, soil microbial communities present a great dynamism so that different species can be temporarily replaced depending on the environmental conditions that soils experience. That dynamism and high diversity of microbial soil communities represent the basis to maintain healthy and well-functioning soil ecosystems. These are important aspects to be considered when analyzing local and global consequences of natural microbial communities in response to the variable environmental conditions existing in our planet. Consequently, the above represent critical factors to understand soil bioremediation processes as a function of environmental parameters and under a current climate change scenario.

### AUTHOR CONTRIBUTIONS

EM executed the experiment in collaboration with EP-F and EG. JG proposed the project and, in collaboration with EP-F and EG, designed and worked out the new methodology. JG prepared the manuscript and provided funding.

### ACKNOWLEDGMENTS

This study was supported through funding from the Spanish Ministry of Economy and Competitiveness (CGL2014- 58762-P; PCIN2016-129) and the Regional Government of Andalusia (RNM2529). These projects have been cofunded by FEDER funds. EM acknowledges funding through a Fullbright fellowship. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

unsaturated porous media – a review. Adv. Water Resourc. 30, 1505–1527. doi: 10.1016/j.advwatres.2006.05.025

Sokal, R. R., and Rohlf, F. J. (1995). Biometry. New York, NY: WH Freeman and Co.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Moxley, Puerta-Fernández, Gómez and Gonzalez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Biological Effects of Thermal Water-Associated Hydrogen Sulfide on Human Airways and Associated Immune Cells: Implications for Respiratory Diseases

Joana Viegas 1 , Ana Filipa Esteves 1 , Elsa M. Cardoso1,2,3, Fernando A. Arosa1,2 , Marco Vitale4,5 and Luís Taborda-Barata1,6,7 \*

1 CICS-UBI–Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal, 2 FCS–Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal, 3 Escola Superior da Saúde, IPG–Instituto Politécnico da Guarda, Guarda, Portugal, 4 DiMeC–Department of Medicine & Surgery, University of Parma, Parma, Italy, 5 FoRST–Fondazione per la Ricerca Scientifica Termale, Rome, Italy, 6 NuESA–Health & Environment Study Group, Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal, 7 Department of Immunoallergology, CHUCB–Cova da Beira University Hospital Centre, Covilhã, Portugal

#### Edited by:

Fabrizio Bianchi, Italian National Research Council, Italy

#### Reviewed by:

Levent Dalar, Istanbul Bilim University, Turkey Yue-Wern Huang, Missouri University of Science and Technology, United States

> \*Correspondence: Luís Taborda-Barata<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Environmental Health, a section of the journal Frontiers in Public Health

Received: 20 February 2019 Accepted: 08 May 2019 Published: 05 June 2019

#### Citation:

Viegas J, Esteves AF, Cardoso EM, Arosa FA, Vitale M and Taborda-Barata L (2019) Biological Effects of Thermal Water-Associated Hydrogen Sulfide on Human Airways and Associated Immune Cells: Implications for Respiratory Diseases. Front. Public Health 7:128. doi: 10.3389/fpubh.2019.00128 Natural mineral (thermal) waters have been used for centuries as treatment for various diseases. However, the scientific background of such therapeutic action is mostly empiric and based on knowledge acquired over time. Among the various types of natural mineral waters, sulfurous thermal waters (STWs) are the most common type in the center of Portugal. STWs are characterized by high pH, poor mineralization, and the presence of several ions and salts, such as bicarbonate, sodium, fluoride, silica, and carbonate. Furthermore, these waters are indicated as a good option for the treatment of various illnesses, namely respiratory diseases (e.g., allergic rhinitis, asthma, and chronic obstructive pulmonary disease). From the sulfide species present in these waters, hydrogen sulfide (H2S) stands out due to its abundance. In healthy conditions, H2S-related enzymes (e.g., cystathionine β-synthase and cystathionine γ-lyase) are expressed in human lungs, where they have mucolytic, antioxidant, anti-inflammatory, and antibacterial roles, thus contributing to airway epithelium homeostasis. These roles occur mainly through S-sulfhydration, a post-translational modification through which H2S is able to change the activity of several targets, such as ion channels, second messengers, proteins, among others. However, in respiratory diseases the metabolism of H2S is altered, which seems to contribute somehow to the respiratory deterioration. Moreover, H2S has been regarded as a good biomarker of airway dysfunction and severity, and can be measured in serum, sputum, and exhaled air. Hence, in this review we will recapitulate the effects of STWs on lung epithelial-immune crosstalk through the action of its main component, H2S.

Keywords: sulfurous thermal waters, hydrogen sulfide, S-sulfhydration, allergic rhinitis, asthma, chronic obstructive pulmonary disease

### INTRODUCTION

Natural mineral waters from thermal springs (thermal waters) are used in Europe since ancient Greece for hygiene and later for the treatment of several diseases (e.g., respiratory, skin, and musculoskeletal diseases). Nowadays these waters are also used beyond their conventional purposes, namely with preventive, anti-stress, and aesthetic functions. The classification of thermal waters (35–40◦C) is based upon their physicochemical features, which allows their subdivision into sulfurous, salso-bromo-iodic, bicarbonate, and bicarbonate-sulfate waters (1, 2). In fact, a beneficial link between sulfurous thermal water (STWs) and clinical improvement of several illnesses has been suggested (3– 6). Such beneficial effects may be due to analgesic, antioxidant (7), antibacterial (8), and anti-inflammatory (9) properties of STWs. Thus, the main advantages of the therapeutic use of STWs lies in the fact that these provide a non-aggressive treatment, without considerable side effects, and which also has preventive properties (6, 8, 10, 11). Nevertheless, knowledge associated with the clinical properties of STWs in the context of respiratory diseases is mainly empiric, acquired over a period of centuries, since few well-designed clinical studies exist. Furthermore, although there are some very interesting in vitro studies on the effects of STWs on cells of the immune system, such studies are scant. Hence, the question of how exactly these waters modulate the observed clinical amelioration is poorly understood.

In this non-systematic review, we will analyze recent and past data obtained from a number of studies that have contributed toward the elucidation of the mechanisms of action of STWs on the lung epithelia-immune interface. To do that, we have performed a compilation of PubMed publications combining the following search terms "sulfurous thermal waters," and "hydrogen sulfide" with the terms "allergic rhinitis," "asthma," "chronic obstructive pulmonary disease" (COPD) "lung," and "lung endothelial cells" with Boolean operator "AND" and "OR." Various combinations were used, in order to focus on specific search questions of the various analyzed topics. No limitations were made in duration of the study or the demographic data of subjects. Literature published in the last 30 years was included. The inclusion criteria in this review were studies conducted with STW, H2S-enriched waters, or H2S for airway diseases, allergic, chronic, rhinosinusitis, COPD, or biological targets and effects of H2S.

The following outcome parameters examined were included in this review: mucocilliary clearance time, nasal respiratory flow and resistance, adverse effects, and immunoglobulin values.

Overall, in the different searches performed, 7,345 studies were obtained. Of these, in total, 7,114 studies were excluded by reading the title or the abstract since they were not relevant or focused on cellular features or disease-related aspects that were not related to the topic of this review. Of the remaining 231 studies, 59 were excluded from analysis because of redundant information of lower quality than that in included studies or because the work focused on issues that were not fully relevant.

### THERMAL WATERS: COMPOSITION AND BIOCHEMICAL PROPERTIES

Depending on their geographical localization, STWs present different physicochemical characteristics. These differences are due to their diverse chemical composition and to the presence of varying amounts of ions and salts, resulting in different therapeutic indications (2, 12).

Portugal is a country with abundant natural mineral (thermal) waters from north to south, as well as in the Portuguese islands, and the frequent visits to bath spas are quite common by the Portuguese population. Among the different types of thermal waters, sulfurous ones are the most common in the north and center of Portugal, with Termas das Caldas da Felgueira (13), Termas de São Pedro do Sul (14), Ferreira et al. (15), and Termas de Unhais da Serra (16) being some of the more representative ones in terms of sulfur-predominant waters. These thermal waters are alkaline (pH = 8.4–8.9), poorly mineralized, and are indicated for the treatment of respiratory, circulatory, digestive, rheumatic and musculoskeletal, as well as metabolic-endocrine diseases (12).

Concerning respiratory diseases, the therapeutic exposure to STWs is performed mostly through inhalation (6, 11, 17), and recently, significant clinical efficacy (e.g., nasal resistance and nasal flow improvement, and reduction of mucocilliary clearance time) was demonstrated when adult and elderly patients underwent hydrogen sulfide (H2S)-enriched nasal water inhalations (18). Inhalator exposure efficacy depends upon various aspects which may affect particle deposition in the airways. These include adopted nebulizer, particle size, airway caliber, and patient's breathing pattern. For instance, the nebulizer must be able to produce particles with a diameter <3µm in order to reach the bronchioli (11). Side effects are another aspect that must always be taken into consideration, whatever the implemented therapy. Even though STWs are generally well-accepted as a safe therapeutic tool due to their low number of side effects (10), these can still occur. In a systematic review and meta-analysis, Keller et al. (19), analyzed all side effects occurring in the pooled total patient population that took part in 13 clinical studies. Focusing on sulfurous waters, after 90 days of STWs treatment, only 19 out of 370 patients presented some adverse events. From those, 13 experienced mild nasal irritations and a sensation of burning, 5 suffered from very limited epistaxis, and one from dermatological hypersensitivity. Moreover, it is of note that even when subjects presented those effects, most of them were local and reversible (19). However, despite common use STWs to achieve a state of well-being and disease amelioration, the cellular and molecular bases underlying these beneficial effects remain unclear.

It was recently found that STWs can induce the production of moderate amounts of neutrophil-attracting chemokines, and low levels of tumor necrosis factor α and interleukin (IL)-6 (9). Even though pro-inflammatory mediators are frequently linked

**Abbreviations:** STWs, Sulfurous thermal waters; H2S, Hydrogen sulfide; IL, Interleukin; COPD, Chronic obstructive pulmonary disease; MAPK, Mitogenactivated protein kinase; NF-κb, Nuclear factor-kappa B; AR, Allergic rhinitis.

to detrimental situations, moderate inflammatory stress may be regarded as positive, according to hormesis theory (20). Thus, mild stress can stimulate body systems repair, in order to prevent further and more severe damage, provided that this state does not involve the accumulation of irreversible changes (20). In addition, with time, inflammation may change the composition of nasal, sinusal, and lung bacterial flora, which may be associated with the development of resistant strains of bacteria (21). This means that inadequately controlled inflammation may lead to bacterial superinfection. Thus, for subjects with inflammatory respiratory disease, which is stable or in its initial phases, STWs may be a good complement to drug therapy since they may contribute toward prevention of recurrent infections caused by various bacteria (11) and viruses (22), and subsequently avoid the progression to a chronic state. Additionally, thermal waters might be also a useful tool in post-operative recovery in cases of chronic rhinosinusitis with or without polyposis, as observed by Staffieri et al. (23). These authors detected a significant reduction of the numbers of inflammatory cells (particularly eosinophils and mast cells) in the nasal mucosa of patients who were treated with sulfurous-arsenical-ferruginous thermal water inhalation after 6 months of having undergone endoscopic sinus surgery (23). Hence, by playing a preventive role in the progression of inflammatory states, STWs may have a prophylactic effect against further inflammation or subsequent superinfection. Furthermore, as a prophylactic agent, STWs may also reduce or even avoid additional hospital costs and degradation of patients' quality of life which are associated with events, such as frequent infection-induced exacerbations or prolonged hospitalizations. Thus, either as prophylactic or therapeutic tools, STWs may not only improve patients' general health and disease-related clinical parameters, but may also show other benefits from a social and financial point of view (e.g., reduction of drug-related costs, decreased hospitalization and disease-specific healthcare costs, national health care decongestion, and a decrease in school and work absenteeism), although some of these benefits were shown in rheumatological diseases and still need to be addressed in costeffectiveness studies of STWs treatments for respiratory diseases (see **Table 1**).

In thermal waters H2S, hydrosulfide ion, and sulfide anion are the most common sulfide species present, with H2S being the most abundant (7).

### BIOLOGICAL PROPERTIES OF HYDROGEN SULFIDE

Due to its inflammable and corrosive nature, H2S was thought to be a poisonous gas. However, in the last decades, it has been reported, along with nitric oxide and carbon monoxide, as a gaseous signaling molecule (25). Indeed, in contrast to nitric oxide, H2S is relatively stable in body fluids, appearing as a promising therapeutic agent in several diseases. Nevertheless, it is important to take into account that H2S can easily pass from water to air (26) and it is a gas which is soluble in water as well as in physiological liquids, and which is volatilized and broken down in vivo (namely in the lungs due to the abundant TABLE 1 | Major beneficial effects induced by sulfurous thermal waters.

STW inhalation can have either a prophylactic or a therapeutic role, which may be associated with potential socio-economic benefits.

presence of oxygen) (27). Thus, H2S frequently mentioned in studies may indicate a mix of H2S and hydrosulfide and sulfide species, alongside with their effects (25, 28, 29).

H2S is a colorless gas with potent reducing properties resulting from geothermal activities (sulfurous mineral water and volcanoes) and found in vegetable proteins (broccoli, garlic), and synthetic compounds (NaHS, GYY4137) (28, 30–34) (**Figure 1A**). In the human body, H2S is produced by a variety of cells (e.g., epithelial, vascular, and smooth muscle cells), and is mainly synthesized from L-cysteine via cytoplasmic and mitochondrial cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) enzymes (30, 35). Endogenous H2S is also generated by the combined action of cysteine aminotransferase, localized in the cytosol and 3 mercaptopyruvate sulfurtransferase, present in the mitochondria (28, 30, 34, 36). Apart from these enzymatic pathways, H2S can also derive from indirect or secondary endogenous sources, particularly from glucose (via glycolysis), glutathione (GSH), inorganic and organic polysulfides, elemental sulfur, and even from bacterial sources present within the gastrointestinal and pulmonary tracts (29, 31, 33) (**Figure 1B**). After H2S synthesis, it can either act on its biological targets or be stored as a bound sulfane sulfur pool, through oxidative formation of hydrodisulfides/persulfides, hydropolysulfides, and polysulfides) (33, 37) as well as an acid-labile sulfur pool, through metalsulfur clusters, free hydrosulfide ion, and persulfides (29, 34) (**Figures 1C,D**). In the presence of oxygen, H2S undergoes oxidation in the mitochondria by sulfide quinone reductase and also via methylation in the cytoplasm, through thiol-Smethyltransferase (30, 38) (**Figure 1E**). In addition, free H2S can be scavenged by methemoglobin and molecules with metal or disulfide bonds (**Figure 1E**), and excreted together with biological fluids (urine and flatus), as well as exhaled in breath (**Figure 1F**) (30, 35).

Due to its high ability to diffuse across lipid membranes without the need of a transporter, H2S can easily reach its molecular targets in a variety of cells, including those in

respiratory, cardiovascular, and neuronal systems (25, 28, 35, 39, 40). Its stability, storage, and release depend upon pH, temperature, and oxygen concentration of the environment. Thus, at physiological pH only a third of total sulfur amount is in the H2S form. At acidic pH, however, H2S is the only form present. In contrast, at an alkaline pH only the bisulfide form exists (28). Oxygen also influences H2S stability since the presence of oxygen induces its conversion to sulfur, which can be further oxidized to hyposulfite, sulfites, and sulfates. In other words, under aerobic conditions H2S is consumed and consequently its effective concentration decreases (29, 41). These findings corroborate the hypothesis that H2S may act as a cellular oxygen sensor (42). Indeed, the decrease in H2S oxidation under hypoxic conditions has been associated with the production of significant amounts of H2S by airway epithelial cells (AECs) (43), akin to production via reduction of preexisting sulfites in mitochondria (44). Fu et al. suggested that this occurs as a result of CSE enzyme translocation to mitochondria, thereby allowing H2S synthesis even after a stress-inducing stimulus, such as hypoxia (45). However, a significant increment of mitochondrial H2S levels and a decrease of its clearance can, therefore, lead to harmful effects, including vasoconstriction and pro-apoptotic effects (46).

The measurement of H2S has turned out to be of major importance to ascertain its putative role in a number of diseases. Although various methods have been used for measuring H2S levels in blood and plasma/serum, such as the methylene blue method, sulfite-sensitive ion selective electrodes, and others, these methods have provided discrepant results, with H2S levels in plasma ranging between 1 and 100µM. This high variability has been attributed to the limitation and lack of sensitivity of the techniques (29, 47), to its capacity to diffuse through cellular membranes, and to the extremely short H2S half-time in vivo (48). Hence, to overcome this obstacle it is mandatory to perform precise biological measurements of H2S.

Regarding the airways, different approaches are used to quantify H2S levels. On the one hand, Saito et al. have suggested that sputum H2S may be a better biomarker than serum H2S in lung-related diseases, since its serum levels may reflect other diseases of peripheral organs (49). Moreover, induced sputum was shown to be an effective sample to assess and identify asthma subtypes (50). However, Saito et al. found a negative correlation between H2S levels in sputum and serum in COPD patients with acute exacerbations (51). This led the authors to propose that an increase in H2S levels in sputum may reflect sequestration of H2S from the circulation into the lungs, with the sputum-to-serum H2S ratio being a good predictor of exacerbations (51). On the other hand, the measurement of exhaled H2S is a non-invasive technique that is not affected by oral conditions (52) and which has been shown to positively correlate with the lung function, particularly forced expiratory volume in 1s (53). Nevertheless, unlike exhaled nitric oxide, H2S levels in exhaled air are not used as an H2S metabolism detector (54). Therefore, either exhaled or sputum H2S could become promising and accurate indicators of airway diseases and airway disease phenotypes.

### Biological Targets of Hydrogen Sulfide

Most of the biological activities of H2S are exerted through protein S-sulfhydration, a post-translational modification process whereas an H2S-derived sulfur group (sulfhydryl) is added to the thiol groups of reactive cysteine residues, originating hydropersulfide (35, 55) (**Figure 2**).

This formed group enhances reactivity of the protein and may be associated with an increment of its biological activity (55). The degree of protein S-sulfhydration can be influenced by cell pH as well as by the distance between the target amino acids and the active core of the protein (35). Unlike S-sulfhydration, S-nitrosylation caused by nitric oxide results in a reduction of cysteine reactivity. As an example, through S-sulfhydration the endothelial nitric oxide synthase (eNOS) activity increases, as a consequence of eNOS dimerization. In contrast, S-nitrosylation

of eNOS induces the formation of eNOS monomers, leading to a reduction of its activity (56).

Thus, H2S is regarded as a gaseous signaling molecule that targets a number of important biological processes and pathways, and in this sense contributes to the maintenance of body homeostasis (35, 57, 58). The biological targets include ion channels, second messengers, signaling molecules, and transcriptions factors. Nevertheless, some of the observed H2S effects are contradictory and probably result from different cell types and concentrations used.

### Ion Channels

H2S is able to interact with many ion channels and, as a consequence, alter their activity by inducing or inactivating their action. It has been reported that by sulfhydrating ATP-activated potassium channels (K+ ATP) in smooth muscle cells (SMCs) (35, 59, 60) and small to medium calcium-dependent potassium channels in vascular endothelial cells (61), H2S can induce the opening of these channels, thereby allowing K+ ion influx, which leads to vasorelaxation.

In contrast, this molecule has the ability to inhibit big conductance calcium-activated potassium channels (35, 57, 61). Moreover, H2S also exerts a regulatory effect on intracellular calcium levels by stimulating or inhibiting the T/L-type calcium channels and in turn may up- or down-regulate several calciumdependent signaling pathways and enzymes, depending upon cell type (35, 61, 62). Furthermore, through the reduction of inositol-1,4,5-triphosphate receptor present in airway SMCs, H2S can also affect calcium efflux in cells (62) (**Figure 3A**).

### Second Messengers

Second messengers are important intracellular signaling molecules involved in several cellular pathways and their availability can be affected by H2S in a direct or indirect way.

Bucci et al. have observed that H2S has the ability to suppress the degradation of cyclic nucleotides by inhibiting the activity of phosphodiesterase, an enzyme that is responsible for their conversion into AMP and GMP (63). Thus, cAMP and cGMP availability increases, making these molecules available for intracellular signal transduction pathways that they are involved in.

The interaction of H2S with calcium ion channels directly affects calcium availability in cells. Therefore, the interaction of this gaseous signaling molecule with calcium ion channels and intracellular calcium pools, can lead to a rise in intracellular calcium by inducing its influx and release, respectively (35). This subsequently induces endothelial proliferation. In parallel, the enhancement of calcium levels can also prompt the translocation of CSE from cytosol to mitochondria, thereby stimulating H2S synthesis inside this organelle, and subsequent mitochondrial ATP production (45). On the other hand, the decrease in intracellular calcium can also be induced by H2S via inhibition of calcium channels, and in turn this suppression seems to be involved in the induction of airway smooth muscle relaxation (64). Moreover, L-type calcium channel inhibition by H2S induces membrane permeability and, subsequently, decreases elastase release (65) (**Figure 3B**).

Recently, it was reported that, under pathological conditions, the S-sulfhydration of the alpha subunit of ATP synthase supports its activation. In this manner, mitochondrial bioenergetics is maintained (66).

### Signaling Molecules

Signaling molecules act as signal transmitters allowing the crosstalk between cells. H2S has also been reported as capable of modulating the activity of several signaling molecules involved in biological processes, including phosphorylation, oxidation and degradation of proteins.

Thus, H2S has been shown to regulate the activity of the mitogen-activated protein kinase (MAPK). In primary human lung endothelial cells, exogenous H2S was able to prevent MAPK activation (67). H2S has also been reported to increase the levels of other kinases, including PI3K/Akt, and protein kinase C, some of which can inhibit the production of pro-inflammatory cytokines (48, 57, 67–69) (**Figure 3C**).

### Transcription Factors

Transcription factors are proteins responsible for regulating genetic information transcription and can also be a target for H2S action. Thus, by undergoing S-sulfhydration transcription factors can induce up- or downregulation of gene expression.

The inactivation of nuclear factor-kappa B (NF-κB) through S-sulfhydration, blocks its translocation to the nucleus, which leads to the suppression of pro-inflammatory mediators and production of adhesion molecules (40). Nonetheless, depending upon the specific inflammatory stage, an opposite effect can be observed (70). Thus, in an initial phase of the inflammatory response, pro-inflammatory cytokine tumor necrosis factor α (TNF-α) can induce CSE transcription, thereby enhancing H2S synthesis. Hence, the newly generated H2S sulfhydrates the p65 subunit of NF-kB, promoting its binding to the coactivator ribosomal protein S3, which results in an increment of antiinflammatory cytokine transforming growth factor-β secretion as well as the stimulation of anti-apoptotic transcriptional activity (57, 70). However, it should be highlighted that in both situations the inflammatory response is diminished, and consequently, oxidative stress decreases.

Moreover, as a consequence of S-sulfhydration of Kelchlike ECH-associated protein 1 (a negative regulator of factor erythroid-related factor 2 activity), the nuclear factor erythroidrelated factor 2 is then translocated to the nucleus. This

results in the expression of antioxidant response elements (68, 71) (**Figure 3D**).

Consequently, S-sulfhydration appears to play an important role in protecting cellular damage, since it has vital implications in anti-inflammatory as well as in antioxidant defenses.

### Biological Effects of Hydrogen Sulfide in the Lung and Associated Immune System Cells

The respiratory mucosal epithelium is the first internal line of defense by acting as a major physical barrier between internal and external environments. The airway surface liquid (comprising mucus and periciliary liquid layers) covers this epithelial surface made up of goblet cells, ciliated cells, basal cells, and club–Clara cells (72–74). It is produced by the epithelial cells, which are also in contact with respiratory gases, thereby constituting the airliquid interface (75). A connective tissue fibroblast-rich layer is located underneath the epithelial surface, and is involved in the maintenance of tissue homeostasis and wound healing (76). This cell layer also contains a variety of cell types, including cells of the innate and adaptive immune system (76). In this layer is located another lung barrier formed by endothelial cells, which separate the bloodstream and vessel walls. The endothelium also has other functions (e.g., blood and oxygen supply, nutrient delivery, and immune cell trafficking) (75, 76).

In physiological conditions, H2S-related enzymes are expressed in the human lungs, namely in SMCs and primary fibroblasts (60, 77). It is now acknowledged that H2S is required for the development of lung vasculature and alveolarisation (78), and in other lung functions, including airway tone and pulmonary circulation (30). Furthermore, H2S appears to be involved in various processes namely airway mucolytic activity, oxidative stress, inflammatory state, cell proliferation, and apoptosis (10, 30). These will be briefly reviewed below (see also **Table 2**).

### Mucolytic Effect

Mucins are secreted by goblet cells, submucosal glands in upper airways (75, 87), and alveolar cells in lower airways (74). By secreting mucins, the mucus traps and absorbs potential harmful pathogens and irritants which are subsequently removed from the respiratory tract by the process of ciliary beating which

#### TABLE 2 | General biological effects of hydrogen sulfide at physiological levels in the lungs.

(Continued)

**20**

mediates appropriate mucociliary clearance (73). An effective removal of mucus protects the respiratory epithelium, acting as a vital role in airways homeostasis (88). Both endogenous and exogenous H2S show positive effects upon the respiratory tract by modulating the mucolytic activity. This appears to result from the interactions between H2S and the disulfide bonds of mucins, resulting in breakage of the latter, which allows the mucus to become less viscous (10). The production of endogenous H2S induces the opening of K+ ATP and the activation of the cAMP pathway (30). Additionally, exogenous H2S inhibits Na +/K+-ATPase and calcium-sensitive potassium channels in human bronchiolar epithelia, thereby triggering electrolyte absorption (33, 89).

This leads to an increase in mucociliary clearance, and therefore the elimination of foreign microorganisms can be more effective (43). Krause et al. also proposed that inhibition of transepithelial sodium absorption (via inhibition of Na+/K+- ATPase) is favored under acute hypoxia, in order to avoid H2S degradation, as well as exogenous H2S exposure (43). Additionally, an amelioration of mucociliary function was observed with inhalation of exogenous H2S, as confirmed by a substantial reduction of mean mucociliary transport time in patients with chronic rhinosinusitis (8, 24).

### Antioxidant Effect

In a well-controlled environment, reactive oxygen species (ROS) are responsible for cell signaling activation, enhancement of pro-inflammatory cytokines acting as mediators of immunity, and in addition, ROS are also able to protect the cells/tissues. Simultaneously, a balance is achieved with a tight regulation performed by antioxidants (e.g., reduced glutathione, GSH) which directly scavenge those species that are formed, thereby inhibiting any excessive production, by removing or repairing cellular damage or modifications induced by these reactive species.

In the lungs, exposure to H2S promotes a boost of antioxidant effects, such as (i) increase in GSH availability, and superoxide dismutase levels (28, 79, 90), and (ii) generation of ATP, replacing oxygen in mitochondrial respiration (91). The reported H2Sinduced mitochondrial protection related to this antioxidant effect is known to result from mitochondrial cytochrome c oxidase inhibition and also from its capacity to modulate cellular respiration, thereby preventing the generation of ROS (48). Consequently, there is an increase in scavenging capacity (30, 34, 92), as well as an induction of endogenous antioxidant defenses (39). Hence, H2S antioxidant features seem to involve both an indirect action and an induction of endogenous antioxidant defenses. For instance, by stimulating cysteine and cysteine transporter activity, H2S induces an augmentation of substrate levels that are necessary for GSH production (29). Unlike large size antioxidants, H2S can easily cross both plasma and mitochondrial membranes. This allows H2S to more promptly reach its biological targets, and therefore it is considered to be more effective at diminishing cellular oxidative stresses, and at increasing antioxidant defenses (80). Braga et al. demonstrated that antioxidant effects of STWs, an exogenous H2S source, provide protection against oxidative DNA damage (93).

and carcinogenic.

### Anti-inflammatory Effect

As the first line in contact with foreign species, AECs can act either as a modulator (during homeostasis) or as a sensor (postinjury lung homeostasis) of innate and adaptive immune systems (74). In healthy states, macrophages are the main immune cell type present in the airways, accounting for about 60– 70% of all cells. Neutrophils represent 30–40% of total cells, while eosinophils are rare, not exceeding 2% (94). Beneath the epithelial layer, dendritic cells, macrophages, and mast cells can be found in the lamina propria. Dendritic cells and macrophages, in particular, can detect pathogens that have crossed the lung epithelium, phagocytose and destroy by microbicidal mechanisms, thereby contributing toward avoidance of significant infections.

Under a normal state the recognition of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) occurs through specific membrane or cytosolic receptors present on epithelial cells, and results in the activation of several signaling pathways (e.g., via MAPK, NF-κB, among others) (95–97). Therefore, pro-inflammatory cytokines, chemokines, antimicrobial proteins, and antiviral substances are produced and released by AECs, which allows them to have a crucial role in the recruitment and activation of innate (e.g., macrophages, eosinophils, dendritic cells) and adaptive (e.g., T and B cells) immune cells (96, 97). The presence of these cells allows several responses (phagocytosis, dendritic cell maturation, chemotaxis, inflammasome activation, and others) to be triggered (97). Subsequently, cells of the adaptive immune system (e.g., T and B cells) are activated. Thus, when homeostasis is affected, the usual function of AECs is reduced and pro-inflammatory activities increase significantly.

The anti-inflammatory properties of H2S can in part be explained by its potent reducing, antioxidant, and scavenging features. Nevertheless, there are controversial results concerning H2S properties, since it appears to exert both pro- and antiinflammatory effects. Some authors have shown an in vitro promotion of granulocyte survival via H2S-induced inhibition of caspase-3-cleavage and p38 phosphorylation (98, 99) and in an oxidative stress environment, activated neutrophils seem to be able to convert H2S into sulfite, which is associated with inflammation (100). Moreover, there are studies suggesting that this gaseous transmitter is involved in GSH depletion and ROS formation (101), and consequently induction of mitochondrial cell death pathways (102). Meanwhile, others have suggested the participation of H2S in some key anti-inflammatory pathways, such as: (i) suppression of leukocyte adherence and migration, mediated by K+ ATP activation in endothelial cells and leukocytes (81), (ii) inhibition of oxidized low-density lipoprotein-induced macrophage inflammation via NF-κB suppression (82), leading to a reduction of several pro-inflammatory cytokines (e.g., IL-1β, IL-6, and IL-8) (103–105), and (iii) reduction of neutrophil toxic effects by inhibiting myeloperoxidase activity (83). However, these discrepancies may be related to differences in H2S concentration. In fact, beneficial effects of H2S generally prevail at lower concentrations, whereas deleterious effects are observed at higher levels and at fast-releasing rates, which is comparable to effects seen with carbon monoxide and cyanide (28, 34, 48). In addition, inhalation of H2S at high acute levels or chronic lower-level exposure can also induce lung inflammation and toxicity (48).

Nonetheless, it has been known for some time that STWs can exert a direct anti-inflammatory effect in the lung. Exposure to these natural mineral waters may induce increased release of IL-10 levels by in vitro exposed primary human monocytes (9). Furthermore, an increase in the levels of IL-10 in saliva were also described in patients who had been treated with STWs (9). Moreover, STWs can simultaneously up-regulate immunoglobulin (Ig) A (an anti-inflammatory immunoglobulin) levels, as well as down-regulate the levels of IgE (a pro-inflammatory immunoglobulin) and cytokines secreted by eosinophils (90). This suggests that by stimulating antiinflammatory defenses, STWs decrease the formation of ROS, and therefore modulate the pro-inflammatory state.

### Antiviral and Antibacterial Effects

In several pulmonary diseases (asthma, COPD, among others) recurrent respiratory tract infections may occur due to a fragile protective mechanism system, which may enhance a proinflammatory state (106). As these diseases progress, viral and bacterial infections may become more frequent, and increasingly more difficult to treat. Consequently, the patients' health state worsens and their quality of life decreases.

Using AECs, Chen et al. shown a link between CSE inhibition and a significant increase in viral replication and chemokine secretion, which are reduced when these cells are exposed to a slow-releasing H2S donor (107). Serum H2S levels are significantly decreased in COPD subjects with very symptomatic exacerbations induced by bacteria and viruses (Type I; Anthonisen criteria), compared to control subjects. However, when COPD exacerbations are less symptomatic (Type II or III; Anthonisen criteria), serum H2S levels are higher than those in control subjects (107). Moreover, when COPD patients who exacerbated are treated with antibiotics, serum H2S levels are lower than in those patients who did not require antibiotic treatment. Taken together, these results indicate that endogenous H2S synthesis is induced in order to counter the infectionsmediated exacerbations (107). Currently, the most frequent therapies used to treat lung viral and bacterial infections are antiviral and antibiotics. However, over time, antibiotic-resistant bacteria may be increasingly observed.

To overcome these problems, some studies focused on the clinical efficacy of therapeutic S-based compounds (e.g., H2S donors, STWs) (22, 84). Previous data showed that these compounds exhibit a protective role as an antiviral agent since they were able to control viral assembly/release (84). In turn, an improvement in viral infection-induced airway hyperresponsiveness can be prompt and may be associated with a decrease in the expression of pro-inflammatory mediators as well as in inflammatory cell influx into the lung (22). Furthermore, a study where patients with chronic sinonasal disease inhaled STWs showed a significant nasal flow improvement in these patients, which was associated with lowering the numbers of nasal bacteria (8). Also, a study in children with frequent

upper respiratory infections showed an important reduction in frequency, duration, severity, and social impact of the infectious episodes with STWs treatment (11). This antibacterial effect may be due to H2S toxicity since only sulfur-bacteria and some microorganisms survive in the presence of S-based compounds, such as those present in STWs (108). This anti-pathogenic role also seems to be linked to the inhibition of bacterial biofilm production, as a result of blockage of the synthesis of microbial adhesins, thereby interfering with bacterial adherence to epithelia and reducing pro-inflammatory potential (6). Also, Varrichio et al. observed a significant reduction of bacteria in children with respiratory infections treated with salso-sulfide thermal water (17). In this context, Benedetti et al. proposed NF-kB pathway as a target of H2S donors to reduce bacteria-induced inflammation (40).

Thus, S-based compounds can be a good complementary approach to conventional drug therapy, thereby increasing clinical efficacy of these treatments.

### Analgesic Effect

Whether and how exactly H2S can modulate an anti-nociception effect remains to be clarified. In patients with perennial allergic rhinitis (AR), a previous study involving inhalation of heated water showed that heat therapy may contribute to relief of perinasal pain and can somehow prevent nasal congestion (109). However, in this study, the heated water was not described in terms of H2S content. Nevertheless, hyperthermia acts as a stimulus of neuroendocrine responses, with an increase in opioid (β-endorphin and Met-encephalin) and adrenocorticotropic hormone levels (110, 111). As mentioned previously, the main compound present in STWs is H2S, and since this therapy is usually performed through inhalation at a temperature between 36 and 38◦C (13, 14, 16), STWs therapy can somehow mediate an analgesic effect in respiratory diseases, mainly in the upper airways, during treatment. This can be explained in part, by the high temperatures of STWs but also by the role of H2S in the activation of K+ ATP. In fact, some studies have suggested that the H2S-mediated analgesic effect is based upon the opening of neuronal K+ ATP, which in turn is related with endogenous opioid system activation (85, 112).

### Anti-proliferative Role

In the lung, cAMP and cGMP are responsible for mediating endothelium-dependent dilatation, since they are involved in lung vascular homeostasis. In fact, a reduction in their levels may lead to pulmonary hypertension, a disease characterized by high blood pressure that affects the lungs' arteries which become narrowed, blocked or destroyed (113). Since H2S inhibits the activity of phosphodiesterase, cGMP net levels increase (63). Therefore, H2S might eventually act as a good auxiliary agent in the treatment of pulmonary hypertension by preventing the proliferation of vascular SMCs and consequently promoting vasodilation of lung blood vessels.

Perry et al. proposed that both endogenous and exogenous H2S might be able to control airway SMCs proliferation and cytokine release, namely IL-8, by suppressing these cell types. This occurs through inhibition of extracellular signal-regulated kinase 1/2 phosphorylation and extracellular signal-regulated kinase-1/2 p38 MAPK, with CBS, and not CSE, the main enzyme involved in the endogenous H2S production (32). Although H2S inhibits the proliferation of SMCs, it promotes the growth of endothelial cells (60, 114).

The inhibitory effect upon cell growth induced by exogenous H2S was also observed by a reduction in lymphocyte subset proliferation, with a subsequent decrease in T cell-derived IL-2 production (115). Similar results were obtained in an in vitro study using STWs (116). This anti-proliferative effect was also shown by Baskar et al. when exogenous H2S exposure caused human lung fibroblast cell death by inducing an increase in DNA lesions in these cells (102). In parallel, cell cycle regulators (p53, p21, and Ku proteins) were activated, being responsible for cell death via apoptosis (102). Therefore, the anti-proliferative effect prevents further degradation of DNA and allows the removal of injured cells by a controlled cell death process. In accordance with this, H2S has been shown to exert a direct suppression effect on human lung fibroblast migration, proliferation, and transdifferentiation into myofibroblasts (117).

### Anticancer Effect

During the last decade, some data have suggested various anticancer effects related to H2S action (118–121). It was proposed that H2S-mediated anticancer role is based upon the combined capacity of H2S to increase the production of metabolic acid lactase and to impair pH regulatory system of cancer cells (86).

However, in contrast, and despite the anticancer efficacy in various types of cancer cells (e.g., gastric carcinoma, renal tumor), other studies have shown that H2S may induce tumor cell proliferation, and migration, as well as angiogenesis, via inhibiting apoptosis (122–124). Jia et al. observed hepatoma cell suppression when CBS/H2S pathway was inhibited (125). Moreover, Szczesny et al. have also found that besides normal lung epithelial cells, epithelial lung adenocarcinoma cells also have the ability to synthesize H2S, in order to maintain their bioenergetics function and to increase its mitochondrial DNA repair processes. In this sense, cancer cell viability was preserved (126).

Hence, in a cancer situation the type of treatment must be re-assessed since H2S seems to be advantageous for cancer cell proliferation. Nevertheless, the H2S-induced inhibitory effects on cancer cell proliferation depend not only on H2S dose and length of exposure, but also on tumor types. In this context, a continuous and prolonged exposure to low H2S concentrations significantly affects cancer cell survival activity (127). In turn, with continuous, high-level exposure to H2S, the cell survival rate is severely affected in both cancer and non-cancer cells (128). Oláh et al. have also indicated varying H2S levels as an explanation for these inconsistent findings regarding anticancer effect of both H2S inhibitors and donors (129). Nevertheless, these authors suggested that if on the one hand optimal endogenous H2S concentration may induce proliferation of cancer cells, on the other hand both endogenous H2S inhibition (decrease of its levels to below optimal concentration) and exogenous delivery (increment of its levels to above optimal

concentration) may suppress cancer cell proliferation (129). Clearly, H2S-mediated effects have to be further studied, namely with various cancer cells lines in vitro as well as in animal models of various cancers, before any firm conclusions can be drawn.

It should also be taken into account that H2S may have a biphasic biological effect, since at low-to-moderate concentrations H2S has a physiological role, whereas at higher concentrations, it exerts a more pathological role, as summarized in **Table 3**.

### Effects of Hydrogen Sulfide in Respiratory Diseases

The human nose and lung are continually exposed to indoor and outdoor agents (e.g., allergens, tobacco smoke, and pathogens). As a result, the airway mucosal epithelium, as an internal line of defense needs to play numerous roles in order to eliminate these foreign agents, thereby allowing normal airway function. On the other hand, after chronic injury and inflammation, the dysregulation of airway epithelial cell function may lead to the pathogenesis of lung diseases, such as AR, asthma, and COPD. These airways diseases are characterized by frequent or persistent respiratory symptoms and intermittent or persistent airflow limitation (130, 131). Moreover, besides the involvement of inflammatory cells and oxidative stress in the pathogenesis of AR, asthma, and COPD, there are various studies suggesting that these patients present alterations in H2S metabolism (132–137).

### Allergic Rhinitis

AR is a chronic inflammatory disease that affects the nasal airways, which become inflamed and engorged after exposure to an allergen to which patients are sensitized. In the nasal mucosa of AR patients, the relevant allergens bind to membrane-bound IgE on mast cells. Cross-linking of various IgE molecules by the allergen induces mast cell activation with rapid release of various pro-inflammatory mediators which are involved in development of acute symptoms. Eosinophils and lymphocytes are also involved, being crucial to the development of chronic symptoms (138, 139). In fact, this inflammatory process disturbs some features of the nasal mucosa, since inflammation is associated with swelling of sinusoidal capacitance vessels, reduction of nasal airway passages size, and an increase in mucus production (140). In addition, nasal airway remodeling may also take place in AR, although whether this really occurs remains controversial. In fact, some studies claim that remodeling is involved, since structural changes (e.g., nasal tissue glandular hypertrophy, collagen or extracellular matrix deposition) were observed, as compared with healthy controls (141, 142). However, in contrast other studies have not found such an association (143, 144).

Park et al. have shown that H2S can be found in human nasal mucosa as well as in the plasma of healthy subjects. This may be partially explained by the presence of CBS and CSE in human nasal epithelium (134). While CBS is mainly distributed in the superficial epithelium and submucosal glands, CSE is exclusively localized in vascular endothelium and surrounding smooth muscles (133, 134). In the nasal and sinus mucosa, the amount of H2S was shown to be increased in mild and moderate/severe persistent AR (134). In this context, there is an enhancement of human H2S-synthesized enzymes, mRNA and protein levels, which consequently leads to a significant increment of H2S levels in human airway (134). This may indicate a compensatory mechanism to attempt to revert the pro-inflammatory state.

Although there are few data specifically regarding STWstreatment of AR, an amelioration in AR patients suffering from allergen-specific non-seasonal rhinitis when treated with a Sbased compound water was observed, and this was associated with a reduction in total IgE and an increase in IgA serum levels (145). In accordance with this, it was suggested that STWs may exert an immunomodulatory activity by inducing an increase in IgA levels in nasal mucus (90). Another study also showed significant amelioration when AR patients were treated with STWs, namely in terms of a significant decrease in nasal flow resistance and nasal mucociliary transport time in 84% of subjects (146). Likewise, a significant reduction of IgE and an increase in IgA levels as well as an improvement of subjective symptomatology assessment scale were also observed (146). Data from these studies suggest a compensatory mechanism in order to reduce the presence of pro-inflammatory mediators and an improvement of the inflammatory state. However, clearly more thorough studies on the immunomodulatory and antiinflammatory effects of STWs treatments in AR patients (as well as in patients with chronic rhinosinusitis) are needed.

Due to common immunopathophysiology, there is a close relationship between AR and asthma ("single airways concept"). In fact, AR is regarded as a risk factor for the development of asthma (147). Thus, although few studies have been carried out with STWs treatment in patients with asthma, we shall now analyse this context.

### Asthma

Asthma is a heterogeneous disease, usually characterized by chronic airway inflammation. Asthma is thus regarded as a long-term inflammatory disorder that results in excessive smooth muscle contraction, hyper-responsiveness, and variable airflow obstruction and bronchospasm. The development and progression of asthma are associated with airway inflammation (inflammatory cells and cytokines), mitochondrial dysfunction, and oxidative stress (148). Excessive airway mucus production is a feature of asthma, particularly in more severe stages, and mucus may even block the airways, which may lead to death by suffocation (149). The difficulty in controlling asthma is partially explained by its clinical and cellular phenotype heterogeneity. Thus, asthma patients may be grouped into different clusters which are associated with preferential features. In this context, one partial but possible subdivision of patients may be into those who preferentially present an eosinophilic bronchial infiltrate and those that have a preferentially neutrophilic profile (150, 151). In addition, it is fundamental to remember that AECs and SMCs are also some of the major cell types involved in asthma pathology, since they are sources of excessive mucus production and the mediators of cell contraction, respectively. This is supported by the underlying episodes of bronchial smooth muscle contraction in asthma patients (152). Likewise, both AECs and SMCs are responsible for facilitating the amplification

TABLE 3 | Dual biological effects of hydrogen sulfide: beneficial vs. deleterious.

H2S, hydrogen sulfide; GSH, glutathione; ROS, reactive oxygen species; K+ ATP, ATP-activated potassium channels.

Exposure to low levels of is associated with beneficial effects. In contrast, exposure to higher levels and fast-releasing rate of exposure to H2S is associated with deleterious effects.

of lung inflammation, by recruiting T cells, which contributes to an uncontrolled pro-inflammatory environment. All of these actions are associated with histological changes in the airways (e.g., increased bronchial wall thickness, mucous metaplasia, and smooth muscle hyperplasia and hypertrophy) which may lead to the impairment of pulmonary function (153).

In human studies, a significant decrease in H2S serum levels was shown in patients with stable asthma, in comparison with healthy subjects. Such a decrease was even more pronounced in asthmatic patients with severe acute exacerbations. It should be pointed out that a similar type of reduction in H2S serum levels was also observed in smokers compared to non-smokers. Moreover, such a decrease was negatively correlated with the percentage of sputum neutrophils in patients with acute asthma (137). Furthermore, both serum H2S levels and lung function parameters were found to be decreased in asthmatic children, suggesting a positive correlation between these two parameters (136). In contrast, Saito et al. found elevated H2S levels in the sputum of asthmatic subjects, but without significant differences between mild and severe stages (49). These authors also observed a positive correlation between sputum H2S levels and sputum neutrophil counts. Nevertheless, serum H2S levels presented a negative correlation with sputum macrophage (49) and eosinophil counts (53).

Previous data have also shown that H2S donors induce vascular smooth muscle relaxation, while they suppress the proliferation of airway SMCs and IL-8 release in humans (32). Since airway SMCs express high amounts of H2S-related enzymes, it appears that the lack of H2S may be involved in the progression and worsening of asthmatic airway obstruction.

However, exposure to environmental H2S levels in excessive concentrations may have deleterious effects in asthma patients. In an epidemiological study carried out in northeast Nebraska (USA), a positive correlation was observed between the frequency of hospital visits by pediatric and adult asthma patients, due to exacerbations, and high (>30 ppb) outdoor total reduced sulfur and/or general H2S levels on the previous day (154). However, it should be noted that actual individual levels of exposure to these sulfur species were not recorded but only inferred. In contrast, Bates et al. did not observe a significant increase in the risk of development of symptoms in asthmatic subjects when exposed to H2S from geothermal activity in New Zealand (155). This suggests that, in certain settings, or in prolonged ambient exposure inhalation of high levels of environmental sulfur species may trigger asthma exacerbations, although this must be studied with more robust study designs and compared with intermittent and controlled exposure which is the situation observed in Spas.

### Chronic Obstructive Pulmonary Disease

COPD is an airways disease caused by significant exposure to noxious particles or gases. The chronic, persistent, airflow limitation associated with COPD is caused by a mixture of small airways disease (e.g., obstructive bronchiolitis) and parenchymal destruction (emphysema) (131). Thus, COPD is characterized by an irreversible persistent decline of airflow associated with an enhanced chronic inflammatory response and emphysematous changes (156). It is well-known that COPD non-smokers and smokers share epithelial susceptibilities features, with direct or indirect cigarette smoking exposure being the main trigger for the development of this disease. In addition, most COPD patients have other chronic co-morbidities, with a proportion of them also having features of asthma–potential Asthma-COPD overlap/ACO (157, 158).

COPD is well-characterized by severe lung structural and functional changes, namely basal, goblet and mucous cell hyperplasia, as well as airway fibrosis (159, 160). Furthermore, a chronic influx of inflammatory cells (T lymphocytes, neutrophils, and alveolar macrophages) in bronchial wall and lumen is also involved in the pathophysiology of the disease (161). As a result of the pro-inflammatory actions, particularly those induced by neutrophil-derived enzymes, the lung parenchyma is destroyed in many cases, leading to emphysema (106, 161, 162).

In a small study performed by Sun et al. using human peripheral lung tissue samples from six patients with COPD, as well as from eleven healthy non-smokers and seven smokers with normal lung function, despite H2S levels being quite similar among the groups, a significant decrease in CSE protein levels and an increase in its mRNA levels was observed in COPD patients as well as in smokers (135). However, in contrast, CBS mRNA levels were reduced in COPD patients as well as in smokers, suggesting that H2S metabolism may be altered in the lung tissue of COPD patients, just as it is in smokers (135). Moreover, the same group showed a negative correlation between exhaled levels of H2S and sputum eosinophilia, in a larger group of 77 COPD patients, suggesting that increased levels of exhaled H2S may predict a non-eosinophilic phenotype in these patients (163). Nevertheless, temporal stability of such phenotypes must be confirmed, since it may vary over time. In fact, Chen et al. also showed that serum H2S levels in COPD subjects vary longitudinally, with higher levels being more frequently seen in a stable state than in acute exacerbations of COPD (132). Both in healthy subjects and in COPD patients with acute exacerbations, H2S levels were more significantly reduced in smokers than in non-smokers. Also, in this study, regarding serum H2S levels, a negative correlation with sputum neutrophils, and a positive association with sputum lymphocytes and macrophages was observed in all patients with COPD. Finally, it seems that H2S availability is also related to the stage of airway obstruction in COPD since its levels are significantly decreased in the more advanced stage III than in the milder stage I (132). Thus, the authors justify the enhancement of serum H2S levels in stable COPD patients as a compensatory mechanism. Nevertheless, increased levels of H2S were observed in COPD subjects with an exacerbation, in comparison with patients with stable COPD, healthy smokers, and non-smokers (51). Despite certain contradictions founded in the previous studies, all indicated significant changes of H2S and H2S-synthesized enzymes levels in COPD subjects. These H2S metabolism alterations seems to contribute, at least in part, to exacerbations and worsening of this respiratory disease state, affecting general lung function.

A significant reduction of oxidative stress and an amelioration of symptoms in subjects suffering from moderate to severe COPD were observed after a 12-day inhalation with STWs and 1 month after the end of the treatment (92). Similar beneficial effects have been observed in patients with chronic rhinosinusitis (6). Further studies focusing on mechanisms underlying such STWs-driven improvement in COPD patients are warranted.

In summary, in spite of generally effective drug-based treatments for most cases of AR, bronchial asthma, and COPD, some patients still show a sub-optimal response to such treatments. Furthermore, over time, long-term high-dose therapy may be associated with the development of some adverse effects. Thus, additional thermal spa complementary therapeutic tool mainly for subjects whose symptoms are not adequately controlled with the usual drug-based therapeutic approach seems to be a good option. With supplementary STWs treatment it may be possible to regain symptom control and eventually reduce baseline drug therapy. Another aspect which must be borne in mind is that with STWs treatment at Spas, additional psychological components may also contribute toward clinical improvement. These components include leisure time, opportunity for relaxation, being aware of regular clinical monitoring, and various cultural aspects, all of which may play a part in Spa treatment-associated final results (164).

### CONCLUSIONS AND FUTURE PERSPECTIVES

Mainly due the presence of H2S, STWs might be an advantageous and promising option as an add-on non-pharmacological complementary therapy for respiratory diseases, such as AR, chronic rhinitis/rhinosinusitis, bronchial asthma and COPD, since these natural mineral waters have been associated with significant, quick onset, and relatively long-lasting improvement of clinical parameters in patients with these diseases. Furthermore, H2S-rich STWs have various effects upon inflammatory and immunological parameters that may contribute toward their clinical efficacy. It may thus be possible to use STWs in a preventive way in terms of disease progression and exacerbations, although this needs to be better ascertained. Moreover, if correctly applied, no significant side effects have been reported with STWs. Nevertheless, in spite of the potential benefits of STWs in respiratory diseases, further elucidation of the mechanisms underlying such benefits warrants further studies. To that end, in vitro models appear to be a promising choice for studying the effects of STWs on human respiratory mucosa. Recently, Epithelix Sàrl (Switzerland) and MatTek Corporation (USA) have developed an in vitro 3D human lung models with a morphologically and functionally differentiated structure as well as an ability to be maintained in a homeostatic state for a long period of time and to be a ready-to-use model (165, 166). In addition, this 3D cell model can also be used to perform safety testing of occupational and environmental chemicals, pharmaceutical development, drug delivery, and inflammatory responses (167–172). Overall, since these in vitro 3D models seem to reproduce human biological responses, they are an interesting choice in the study of various respiratory diseases, such as AR, asthma, and COPD, namely in terms of their response to STWs exposure, and this is one of the current interests of our research, which is in accord with current ethical concerns regarding the use of animals in research investigations, i.e., the 3R (Replace, Reduce, Refine) and animal welfare.

### CONTRIBUTION OF THIS REVIEW TO THE FIELD

This review is novel andignificantly contributes toward having a clearer and more organized view of the role of sulfurous thermal/natural mineral waters (STWs), in inhalational treatment of respiratory diseases, such as rhinitis, asthma or chronic obstructive pulmonary disease. Furthermore, this review also recapitulates the main effects of STWs on lung epithelialimmune crosstalk through the action of its main component, H2S, thereby establishing a relationship between clinical efficacy of these treatments and the underlying immunopathological and immunotherapeutic mechanisms.

### AUTHOR CONTRIBUTIONS

JV and AE carried out literature searches and wrote the manuscript. EC checked the biochemical aspects. FA checked the immunological aspects. MV and LT-B checked the clinical aspects. All authors made contributions to the

### REFERENCES

review. EC, FA, MV, and LT-B checked the final version of the manuscript.

### FUNDING

This work was supported by CENTRO 04-3928-FEDER-000010, financed by PROVERE, CENTRO 2020, Portugal 2020, and FEDER and National Funds by FCT–Foundation for Science and Technology (Project UID/Mul]/00709).

effects of sulphurous thermal water inhalation. Sci World J. (2013) 2013:7. doi: 10.1155/2013/927835

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Viegas, Esteves, Cardoso, Arosa, Vitale and Taborda-Barata. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Visitors to a Tropical Marine Beach Show Evidence of Immunoconversions to Multiple Waterborne Pathogens

Kaneatra J. Simmons 1†, Tarsha N. Eason2 , Clarissa L. Curioso3 , Shannon M. Griffin4 , Malini K. D. Ramudit 3 , Kevin H. Oshima4 , Elizabeth A. Sams 5 , Timothy J. Wade5 , Ann Grimm4 , Alfred Dufour 4 and Swinburne A. J. Augustine4 \* †

*1 Department of Arts & Sciences/Learning Support, Fort Valley State University, Fort Valley, GA, United States, 2 National Risk Management Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH, United States, 3 Oak Ridge Institute for Science Education, Oak Ridge, TN, United States, 4 National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH, United States, 5 National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC, United States*

### Edited by:

*Ethel Eljarrat, Institute of Environmental Assessment and Water Research (CSIC), Spain*

#### Reviewed by:

*Jill Stewart, University of North Carolina at Chapel Hill, United States M. Jahangir Alam, University of Houston, United States*

#### \*Correspondence:

*Swinburne A. J. Augustine<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Environmental Health, a section of the journal Frontiers in Public Health*

Received: *10 June 2019* Accepted: *02 August 2019* Published: *19 August 2019*

#### Citation:

*Simmons KJ, Eason TN, Curioso CL, Griffin SM, Ramudit MKD, Oshima KH, Sams EA, Wade TJ, Grimm A, Dufour A and Augustine SAJ (2019) Visitors to a Tropical Marine Beach Show Evidence of Immunoconversions to Multiple Waterborne Pathogens. Front. Public Health 7:231. doi: 10.3389/fpubh.2019.00231* Determining infections from environmental exposures, particularly from waterborne pathogens is a challenging proposition. The study design must be rigorous and account for numerous factors including study population selection, sample collection, storage, and processing, as well as data processing and analysis. These challenges are magnified when it is suspected that individuals may potentially be infected by multiple pathogens at the same time. Previous work demonstrated the effectiveness of a salivary antibody multiplex immunoassay in detecting the prevalence of immunoglobulin G (IgG) antibodies to multiple waterborne pathogens and helped identify asymptomatic norovirus infections in visitors to Boquerón Beach, Puerto Rico. In this study, we applied the immunoassay to three serially collected samples from study participants within the same population to assess immunoconversions (incident infections) to six waterborne pathogens: *Helicobacter pylori, Campylobacter jejuni, Toxoplasma gondii,* hepatitis A virus, and noroviruses GI. I and GII.4. Further, we examined the impact of sampling on the detection of immunoconversions by comparing the traditional immunoconversion definition based on two samples to criteria developed to capture trends in three sequential samples collected from study participants. The expansion to three samples makes it possible to capture the IgG antibody responses within the survey population to more accurately assess the frequency of immunoconversions to target pathogens. Based on the criteria developed, results showed that when only two samples from each participant were used in the analysis, 25.9% of the beachgoers immunoconverted to at least one pathogen; however, the addition of the third sample reduced immunoconversions to 6.5%. Of these incident infections, the highest levels were to noroviruses followed by *T. gondii.* Moreover, many individuals displayed evidence of immunoconversions to multiple pathogens. This study suggests that detection of simultaneous infections is possible, with far reaching consequences for the population. The results may lead to further studies to understand the complex interactions that

**32**

occur within the body as the immune system attempts to ward off these infections. Such an approach is critical to our understanding of medically important synergistic or antagonistic interactions and may provide valuable and critical information to public health officials, water treatment personnel, and environmental managers.

Keywords: immunoconversions, incident infections, exposure, multiplex immunoassay, salivary antibodies, antibody response, Luminex, Boquerón Beach

### INTRODUCTION

Understanding the complex relationship between water quality, human exposure to waterborne pathogens, and subsequent infections is an ongoing public health challenge. Previous studies have shown considerable risk of illness related to polluted recreational and potable water (1–4). One of the primary risks associated with such exposures is the manifestation of acute gastrointestinal illness which may be caused by a variety of viruses, bacteria, and protozoa. In lieu of classic approaches to studying exposure (e.g., water sampling, risk modeling, epidemiological surveys, enzyme-linked immunosorbent assays), we developed a bead-based salivary IgG antibody multiplex immunoassay using the LuminexTM xMAP platform. Many traditional immunological studies involve examining antibodies in a monoplex, one pathogen at a time. Monoplex immunoassays, in general, do not truly capture the dynamic nature of the immune system and obscure our ability to observe simultaneous antibody responses to multiple pathogens. The multiplex developed and applied in this study assesses IgG antibodies in saliva to examine exposure to multiple pathogens simultaneously (5–7).

Saliva is a biofluid with emerging applications in research and clinical settings. We previously demonstrated that salivary IgG antibodies offer a viable alternative to the traditional use of blood, serum, and plasma for assessing immunoprevalence to waterborne pathogens (5, 8, 9). Researchers have noted the many benefits of using saliva including ease of collection, transport, and storage, applicability to remote collection and point-of-care settings, non-invasiveness, and the use of sampling mechanisms which are easily tolerable by children. The use of assays based on saliva are thus expected to have positive impacts on infant and child health (10–12).

We previously determined the prevalence of salivary antibodies against six waterborne pathogens: Helicobacter pylori, Campylobacter jejuni, Toxoplasma gondii, hepatitis A virus, and norovirus genogroups G.I.I and GII.4 in beachgoers and swimmers at Boquerón Beach, Puerto Rico using the multiplex immunoassay (5–7, 13, 14). We also linked epidemiological factors to the norovirus exposures and were able to identify asymptomatic infections (i.e., detectable infections unlinked to symptomology) associated with swimming at a fecally contaminated beach (15). As a follow-up, we aim to determine immunoconversions and evidence of multiple infections to the six target pathogens using samples collected from visitors to the beach which has been shown to be impacted by sewage from publicly-owned treatment works.

Immunoconversion relates to the development of detectable antibodies in the blood and saliva, usually within days after exposure to infectious agents. Soon after an initial exposure and during the acute phase of an infection, IgM antibodies along with other immune system components dominate the response. Later IgG antibodies that retain memory of the exposure are produced and these immunoglobulins rapidly expand in the event of a secondary infection as part of what has been described as the anamnestic response (16).

Previous immunoconversion studies relied on two serum samples (one pre-exposure and another post-exposure) obtained via invasive procedures. These studies measured a 4-fold increase in antibody response from the first to second sample to determine whether there was a change in antibody levels indicative of an active infection. In our study, we collected an initial sample (S1) at the beach before swimming, and the remaining samples (S2 and S3) were self-collected by participants 10–14 and 30– 40 days later, respectively. After collecting and processing the samples, we compared immunoconversion rates given traditional criteria based on two samples to the rates using criteria based on all three-samples. Further, we explored the data to assess whether there was evidence of simultaneous co-infections (immunoconversions to two or more pathogens). Co-infections have been shown to aggravate and exacerbate the symptoms of AGI-related diarrhea (17, 18).

### MATERIALS AND METHODS

### Reagents

5.6 micron polystyrene microsphere (bead) sets were obtained from Luminex Corp. (Austin, TX, USA) at a concentration of 12.5 × 106 beads/ ml each. Biotinylated goat anti-human IgG (λ) secondary detection antibody was obtained from KPL (Gaithersburg, MD, USA). Antigens were purchased as described and coupled to the beads in accordance with the optimized multiplex immunoassay (5, 8). Characterized sera was obtained from SeraCare (Milford, MA, USA) to validate the assays.

### Antigen Coupling and Confirmation Using Animal-Derived Antibodies

Beads were activated and coupled, as previously described (5, 8) and serial dilutions of primary capture antibodies specific to each antigen were used to confirm that the beads were coupled properly thus ensuring that the dynamic range of the assay could be defined (5, 8). Briefly, coupled bead stocks were diluted in PBS-1% BSA to a final concentration of 100 beads/µl of each unique bead set. 5 × 103 beads from each bead set was added to individual wells of a pre-wet 96-well filter plate. An equal volume 2-fold serial dilutions of anti-species IgG primary antibody was added to the beads, mixed gently, covered, and allowed to incubate in the dark, at room temperature for 30 min at 500 rpm on a VWRTM microplate shaker (Radnor, PA, USA).

After incubation, supernatant was vacuumed out, wells were washed twice with 100 µl of PBS pH 7.4 containing 0.05% Tween 20 (PBS-T) (Sigma, St. Louis, MO, USA) and vacuumed again to remove excess buffer. Beads were resuspended in PBS-1% BSA buffer and exposed to 0.8 µg of biotinylated anti-species IgG secondary detection antibody. The filter plates were covered and allowed to incubate in the dark at room temperature for 30 min on a plate shaker. After a 30-min incubation in the dark on a plate shaker to protect the beads from bleaching, the wells were washed twice as above. Then the samples were incubated for 30 min with 1.2 µg of streptavidin-R-phycoerythrin, vacuumed, washed X2, and resuspended in 100 µl of PBS-1% BSA. The plates were then analyzed on a Luminex 100 analyzer (Luminex Corporation, Austin, TX, USA).

### Saliva Collection, Processing, and Analysis

Saliva samples were collected from 2,091 study participants at Boquerón Beach, Puerto Rico during the summer of 2009 as part of the National Epidemiologic and Environmental Assessment of Recreational Water Study (NEEAR) conducted by the United States Environmental Protection Agency (19). A key criterion for selecting the beach was ensuring that local residents were the primary visitors; hence, while tourists do recreate at the beach, visitors are largely locals with over 70% indicating six or more visits to the beach annually. Informed consent was obtained from subjects in accordance with Institutional Review Board approval (IRB # 08-1844, University of North Carolina, Chapel Hill, NC, USA). Study participants were instructed to rub the OracolTM saliva collection device (Malvern Medical Developments, Worcester, U.K) against the gingival crevices of the oral mucosa (between the gums and teeth) to absorb saliva. Infants younger than 1 year old were excluded from the study because of the potential for contamination by maternal antibodies and high rates of non-waterborne infections. Individuals who reported dental or other illnesses at the time of the initial collection were also excluded.

The baseline samples (S1) saliva samples were collected on the beach by trained study staff members while the second and third samples were self-collected by the participants at home and within a day or two post-collection, the samples were shipped overnight on ice to USEPA in Cincinnati for storage and analysis. Upon receipt, the swabs were stored at −80◦C until ready for processing which typically occurred in <1 week. The OracolTM saliva collection devices were thawed to room temperature and centrifuged twice (first at 491 × g, 10◦C for 5 min to recover the saliva off the collection sponge and then at 1,363 × g, 10◦C for an additional 5 min to pellet debris from the saliva) and transferred to 1.5 ml microcentrifuge tubes. Finally, the samples were centrifuged at 1,500 × g for 3 min and the supernatant transferred to a fresh 1.5 ml microcentrifuge tube and stored at −80◦C.

A 1:4 dilution of the saliva samples in phosphate buffered saline containing 1% bovine serum albumin (PBS-1% BSA) was added to prewet and vacuumed 96-well filter plates (Millipore, Billerica, MA, USA). 5 × 103 beads from each bead set and an equal volume of diluted saliva were loaded onto each well resulting in a final dilution of 1:8 in a total volume of 100 µl per well. The loaded filter plates were processed, as previously described (5, 8), reporter fluorescence was measured using a Luminex 100 analyzer and expressed as MFI (Median Fluorescence Intensity) of at least 100 beads per bead set.

### Assay Controls, Cross-Reactivity, and Signal to Noise Ratio (SNR)

Assay controls are described in greater detail in previous publications. Briefly, an uncoupled unique bead set was added to the assay to evaluate non-specific binding and sample to sample variability (5, 8). These control beads were treated identically to antigen conjugated beads and blocked with BSA. However, they were not coupled to any antigen during the coupling step. To control for non-specific binding and/or contamination of the saliva by serum from gum disease or other sources, samples with reactivity to uncoupled control beads at ≥500 MFI were discarded.

Tests for cross-reactivity were performed in monoplex, duplex, and multiplex for each antigen. Assay sensitivity was validated with characterized human plasma samples as previously described (8). A signal to noise ratio (SNR) was calculated by dividing the MFI of the specific antigen signals by the MFI of the uncoupled control beads for each sample (8, 20).

### Defining Immunoconversions and Data Analyses

Only study participants who gave all three samples were considered and two definitions, or immunoconversion criteria (IC) were evaluated. Criteria A is based on the commonly used approach for determining seroconversions for clinical samples collected in a controlled environment: a 4-fold MFI increase from S1 to S2 (S2 ≥ 4 × S1) (14, 21, 22). The second criterion (Criteria B) was developed by extending the traditional definition to ensure that the S2 sample MFI is immunopositive [greater than the cut-off point (9)]; and the S3 sample MFI is at least three times that of the S1 sample (S2 ≥ 4 × S1; S2 ≥ cutoff; S3 ≥ 3 × S1). To reduce potential false positives, we measured antibody responses in the S3 sample. IgG levels are expected to remain relatively high and not drop to zero during the 30–40 day period after initial exposure for all the organisms in this assay. The addition of the S3 sample may guard against considerable variation in volume and composition as well as possible errors in labeling since S2 and S3 were self-collected. All data analyses were performed using Microsoft Excel 2016, JMP 14, and MATLAB Release 2018b.

### RESULTS

### Study Population

Study participants provided 5,533 serially collected samples which were processed to prepare the immunoassay results for statistical analyses (**Supplementary Material**). Before assessing immunoconversions, we removed samples with control bead MFI ≥500 (as previously noted) and computed a mean MFI of samples with duplicate IDs. The remaining samples included saliva collected from 2,078 individuals at the beach (S1) and then self-collected by 1,694 individuals at S2 and 1,666 individuals at S3. Scatterplots of the MFI results suggest that IgG antibodies to the noroviruses were more prevalent (the most frequently detected of the targeted pathogens) in the study population followed by hepatitis A virus (HAV) and H. pylori (**Figure 1**). In contrast, most of the T. gondii and C. jejuni MFI results were near the level of the control beads. Of these samples, MFI results were available from 1,399 individuals who provided saliva during all three collection periods. This cohort was used to estimate the number of immunoconversions by measuring IgG antibody levels against the six antigens in the Luminex multiplex immunoassay and computing immunoconversions based on the criteria developed.

### Immunopositivity and Immunoconversions

A cut-off value of 505 [criteria = 10mean(h)+3SD(h), where h = log (control) and SD = standard deviation] was previously developed as a part of the Boquerón immunoprevalence study (9); hence, samples with MFI ≥505 for the study pathogens are considered immunopositive. A heatmap for the target pathogens (**Figure 2**) provides a visualization of the immunopositive samples (in red) at each collection time point (**Figure 2**, upper panel). Results indicate that over 60% (68, 61, and 62% in S1, S2, and S3, respectively) of the individuals were immunopositive to at least one pathogen during each sample period. As expected, the cohort of beachgoers was primarily exposed to noroviruses (NoV GI.1: 45.34% and NoV GII.4: 30.05%) followed by HAV (15.68%; reported as a mean over the sample periods) and the highest levels were found in the baseline sample (S1), indicating that these participants were exposed prior to providing the initial sample on the beach.

MFI trends reflect antibody response patterns and afford the ability to estimate immunoconversions in the study population. When comparing the two immunoconversion definitions, there were 363 (25.9%) and 91 (6.5%) of the 1,399 individuals who immunoconverted to at least one pathogen, given Criteria A and Criteria B, respectively. For Criteria A, the immunoconversions amongst the specific pathogens ranged from 97 (6.93%) for NoV GII.4 to 166 (11.87%) for T. gondii. Conversely, there were much fewer immunoconversions based on Criteria B with most being to noroviruses (NoV GI.1: 39(2.79%); NoV GII.4: 36(2.57%) followed by T. gondii (22(1.57%) (**Table 1**). While most of the immunoconversions were to one pathogen, many individuals immunoconverted to multiple simultaneously (Criteria A: 46.56 %; Criteria B: 24.18 %).

Results from the less stringent criteria (Criteria A) reflected multiple simultaneous immunoconversions to all the pathogens in the study. Although several of the samples were found to meet the 4-fold increase guideline between S1 and S2, many of these samples never exceeded the immunopositivity cut-off level (MFI ≥505). Therefore, these samples were considered immunonegative. Accordingly, we shifted away from Criteria A and used the more stringent definition (Criteria B) which not only assures that the second sample is immunopositive, but by including S3, also affords the ability to capture antibody patterns that mimic the anamnestic response and could help reduce the number of false positives.

Using Criteria B to further examine immunoconversions, we note that 69 individuals (75.82%) immunoconverted to one pathogen and 22 individuals immunoconverted to multiple (24.18%), resulting in 120 total immunoconversions. **Figure 3** provides IgG antibody response curves for the immunoconversions captured during the study period. In the plots, the dashed red line represents the immunopositivity cut-off value (MFI = 505) and helps to highlight the number of ways individuals immunoconverted to the target pathogens based on Criteria B. All immunoconversions met the core criteria (S2 ≥ 4 × S1; S2 ≥ cutoff; S3 ≥ 3×S1), yet a small number of them (n = 9) presented with immunopositive baseline samples indicating previous exposures, mostly to noroviruses. In addition, rather than declining from S2 to S3, some of the MFIs continued to increase suggesting the possibility of new exposures at S2. Of the immunoconversions to multiple pathogens, the majority (n = 18) were to only two pathogens. **Figure 4** provides a breakdown of the co-immunoconversions observed by specific pathogen pairs. Nearly half of these co-immunoconversions were to the noroviruses (44.4%) and over a fifth were to H. pylori and Nov GI.1 (22.2%) and H. pylori and hepatitis A (22.2%).

### DISCUSSION

Detecting exposure and incident infections to environmental pathogens has long been a complex environmental and public health challenge. Classic approaches involve epidemiological studies and surveys capturing demographic and symptomology information, collection of water, soil, and/or air samples to detect organisms in media and complex modeling to estimate potential impacts on human populations. As an augmentation of this practice, this effort extends the use of salivary antibodies as biomarkers of exposure. Rather than estimating the risk of exposure, our approach relies on the body's immunological response to determine whether exposure and subsequent infection to specific pathogens has occurred. Capitalizing on the availability of antibodies in saliva samples, we developed a multiplex immunoassay to measure IgG antibodies against waterborne pathogens. The optimized assay was used to estimate immunoprevalence to targeted pathogens given saliva samples collected from beachgoers at Boquerón Beach, Puerto Rico (9). Additionally, we identified a breakthrough connection between asymptomatic norovirus infections and swimming at the beach (impacted by sewage from publicly-owned treatment works) finding that infected parties did not necessarily present with gastrointestinal symptoms (15). The assay was also applied in a prospective community study of waterborne infections to characterize associations between water-related exposures, Cryptosporidium and norovirus infections and gastrointestinal symptoms (6).

In this work, we applied the multiplex immunoassay to investigate antibody responses and detect evidence of immunoconversions to six target pathogens in visitors at

Boquerón beach, Puerto Rico. Our effort was a blind examination of the study population focused on IgG antibodies characterized by patterns in median fluorescence intensity and aimed at providing an immunological response screening approach for all pathogens in the multiplex.

After applying the immunoassay and processing the MFI results, we found that 1,399 visitors provided samples during all three collection periods and on average, 892 (64%) of them displayed evidence of detectable antibodies to at least one pathogen (9). This is consistent with previous studies of acute gastrointestinal illness (AGI) performed in multiplex (23– 26). These observations demonstrate the potential for multiplex assays to identify multiple infections and may be applied either cross-sectionally or longitudinally to provide insight into the epidemiology of these conditions, as well as understanding the role of potential risk factors.