Impact of Air Pollution on the Composition and Diversity of Human Gut Microbiota in General and Vulnerable Populations: A Systematic Review

Recently, growing attention has focused on the impact of air pollution on gut microbiota as a possible mechanism by which air pollutant exposure increased the risk for chronic diseases, as evidenced by in vivo studies demonstrating important exposure-induced alterations in the diversity and relative abundance of gut bacterial taxa. This systematic review provides updated state-of-art findings of studies examining the impact of air pollution on the human gut microbiota. Databases PubMed, Scopus, and Web of Science were searched with the following strategy: “air poll*” AND “gut micro*” OR “intestinal micro*”; moreover, a total of 10 studies were included. Overall, there is the evidence that short-term and long-term exposure to air pollutants have the potential to alter the composition and diversity of gut microbiota; some studies also correlated air pollution exposure to adverse health effects (impaired fasting glucose, adverse pregnancy outcomes, and asthma attacks) via alterations in the composition and/or function of the gut microbiota. However, the evidence on this topic is still scarce, and large cohort studies are needed globally.


Introduction
Air pollution represents one of the most important environmental threats to public health globally, and it is responsible for an increased risk of morbidity and mortality due to cardiopulmonary and neoplastic diseases [1,2]. Epidemiological studies have shown an association between exposure to air pollutants and an increased incidence of asthma and chronic obstructive pulmonary disease [3][4][5]; their exposure has also been associated with the development of lung cancer [6][7][8]. Lastly, exposure to air pollution has been linked with an increased risk of stroke and ischemic heart disease [9,10].
According to the World Health Organization, around 90% of individuals globally, living in urban areas, are exposed to air containing high levels of pollutants, and about 4.2 million people die prematurely from outdoor air pollution each year [2,11]. In urban areas, rapid economic development and urbanization have concurrently boosted energy consumption and pollutant emissions, which has led to increasingly serious environmental pollution [12]. The air pollutants include gaseous components (carbon oxides, nitrogen oxides, sulphur oxides, and ozone), volatile organic compounds (e.g., hydrocarbons also halogenated), and fine and ultrafine particulate matter (PM) that may contain metals, semi-metals, and persistent organic pollutants. All these contaminants may determine deleterious effects on human health [13].
Evidence has been provided on air pollutant's ability to directly irritate airway epithelium and cause oxidative stress and inflammation, underlying the development or progression of respiratory diseases, including lung cancer [14,15]. In addition, air pollutants may reach the gastrointestinal tract through the muco-ciliary clearance of inhaled pollutants or the intake of contaminated food and water [14]. Once in the gastrointestinal tract, air pollutants may interact with the intestinal epithelium, promoting the production of reactive oxygen species and pro-inflammatory oxidative lipids and contributing to the development of intestinal diseases [16,17]. In support of this hypothesis, recent studies have associated air pollution with intestinal diseases [13], including colorectal cancer [18] and inflammatory bowel diseases [19].
More recently, growing attention has focused on the impact of air pollution on gut microbiota as a possible mechanism by which air pollutant exposure increases the risk for chronic diseases. In this regard, particularly interesting are in-vivo studies demonstrating important exposure-induced alterations in the diversity and relative abundance of gut bacterial taxa [20][21][22].
Intestinal microbiota is more complex than other microbiota niches [23][24][25], for instance, the genital microbiota [26,27], and accounts for approximately 10 13 bacteria; the most dominant bacterial phyla are Firmicutes (60-80%) and Bacteroidetes (20-40%), while Proteobacteria, Actinobacteria, Fusobacteria, and Verrucomicrobia are in the minority. Typically, obligate anaerobes (e.g., Bacteroides, Clostridium, Fusobacterium, and Bifidobacterium) prevail over facultative anaerobes (e.g., Lactobacillus, Escherichia, and Enterococcus) and the balance of the intestinal microbiota is essential for the integrity of the intestinal epithelial barrier and its functions as well as for the protection against pathogenic challenges. On the contrary, a disbalance of the gut microbiota composition, namely dysbiosis, has been associated with several chronic conditions, such as inflammatory bowel disease [28], cancer [29], and metabolic disorders [30].
The intestinal microbiota composition is highly variable among individuals; specifically, the abundance of specific bacteria varies in relation to several factors, including age, diet, the administration of antibiotics, and, as recently highlighted, environmental pollution exposure [31].
This systematic review provides updated state-of-art findings of studies examining the impact of air pollution on the human gut microbiota. Potential mechanisms by which air pollution can cause adverse health effects via the alteration of human microbiota are also described.

Selection Protocol and Search Strategy
This systematic review was registered in the International Prospective Register of Systematic Reviews, registration number: CRD42022328390. The protocol was written according to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statement [32]. Zotero citation management software (RRID: SCR_013784) was used to identify any duplicates and to manage and screen the selected literature records.
The research study was performed between 23rd and 31st May 2022 in databases PubMed, Web of Science, and Scopus. We restricted the publication language to English. Relevant studies were identified using the following keyword and Boolean operator combinations: "air poll*" AND "gut micro*" OR "intestinal micro*". Truncation filters (*) were used to represent any combination of letters. Three independent reviewers (SF, MDP, and CP) performed the search, reading the titles and abstracts of the articles identified by the search strategy. During the multi-step exclusion process, disagreements on the studies were discussed until reaching a consensus. The process was supervised by other investigators (MV and RS). The PRISMA flow chart diagram was used for summarizing the selection steps for the present systematic review.

Study Selection and Eligibility Criteria
Three (SF, MDP, and CP) of the co-authors independently performed study selection using the Population, Intervention, Comparison, Outcomes, and Study (PICOS) criteria for inclusion and exclusion criteria. We included all studies meeting the following criteria: (1) all genders and all age individuals; (2) exposure to ambient air pollution; (3) gut dysbiosis outcome. Exclusion criteria were as follows: (1) exposure to indoor air pollution; (2) respiratory outcomes and skin microbiota dysbiosis. Animal studies, reviews, systematic reviews, meta-analysis, editorials, commentaries, case reports, case series, semi-experimental and experimental studies, proceedings, individual contributions (e.g., conference speeches), and purely descriptive studies published in scientific conferences without any quantitative or qualitative findings were excluded from the review.

Data Extraction Process and Quality Assessment
Three authors (SF, MDP, and CP) independently extracted the following information from each included study: first author's name, publication year, study region, study period, study population, sample size, study design, parameters of air pollution, duration of exposure, type of exposure, exposure assessment methods, and confounding factors. Differences in the composition and diversity of gut microbiota and any measure of association to air pollutants were reported as main results. Two different reviewers (MV and RS) assessed the methodological quality of the selected studies with the Newcastle-Ottawa Scale (NOS) rating tool, adapted for evaluating case-control, cross-sectional, and cohort studies [33]. The NOS is divided in eight categories evaluating three different quality aspects: selection, comparability, and outcome; scores range from 0 to 9, and the quality of a study was high if the NOS score was 7 to 9, intermediate if the NOS score was 4 to 6, and low if it was 0 to 3 [34].
Studies were also assessed according to the "Strengthening The Organization and Reporting of Microbiome Studies" (STORMS) checklist criteria for reporting metagenomic studies [35].

Study Selection and Characteristics
After searching the designated databases, 511 articles were retrieved. A total of 196 articles were excluded after removing duplicates. Out of the remaining 315 papers, 154 were subjected to further screening and evaluated for inclusion in the systematic review after considering inclusion and exclusion criteria. Finally, 12 articles were considered eligible to be included in the systematic review, but 2 articles were excluded for missing data. A total of 10 articles were used for the data extraction [36][37][38][39][40][41][42][43][44][45]. The PRISMA flow diagram in Figure 1 illustrates the search results. Tables 1 and 2 described the characteristics of the studies included in the systematic review. The included studies (nine cross-sectional and one cohort study) were published from 2018 to 2022 [36][37][38][39][40][41][42][43][44][45] (Table 2). Among the ten studies, two of them were conducted in America [39,45], seven in China [36][37][38][40][41][42][43], and one in Europe (Finland) [44], and the sample size was very variable, ranging from 21 to 6627 subjects.
The studies included healthy individuals as well as vulnerable populations such as obese adolescents, asthmatic children, schizophrenic patients, and pregnant women with adverse pregnancy outcomes. Both male and female were enrolled in all studies, with the exception of one study that included females only [38]. Children, adolescents, young adults, adults, and older people were included, with ages ranging from 5 to over 70 years.
As for the STORMS metagenomic checklist, all included studies provided a partial description of methods for sample collection, storage, and DNA isolation. Sequencing methods were reported by almost all included studies. 16S rDNA gene sequencing was performed in nine studies by using Illumina Hiseq or Miseq platforms, whereas wholegenome shotgun sequencing was conducted in one study [39] via the Illumina Hiseq platform. The region of amplification of the 16S rDNA gene varied among studies, such as V4 (n = 4), V3-V4 (n = 2), and V5 (n = 1) ( Table 2). Two studies mentioned that they used three different regions (V4, V3-4, and V4-5), but it was unclear how these analyses were integrated into bacteria characterization [37,42]. Moreover, the primers used for the amplification varied across studies: three studies used 515F/806R primers, and two used 341F/805R and 338F/806R, respectively ( Table 2).
The majority of the studies (n = 8) reported quantitative measures of microbial diversity and richness within bacterial community via different alpha-diversity indices (e.g., Chao1's, Shannon's, and Simpson's indices). Two studies reported a β-diversity measure [41,42] to evaluate differences in microbial abundances between multiple samples. Lastly, three studies performed linear discriminant analyses for identifying potential markers of dysbiosis associated with air pollution exposure [38,40,41]. Functional gene pathways were investigated in two studies [39,41]. Concerning the study quality assessment, the median NOS score was 6 (interquartile range 1.75), hence indicating an intermediate average level. Specifically, n = 4 studies were of high quality (NOS equal to 7), and n = 6 studies were of intermediate quality (score of 5 to 6). Table S1 shows the results of the scoring method applied to each study included in the review, with reference to publication year.
Overall, a significant correlation between exposure to specific air pollutant and alteration in human gut microbiota was found, and it did not depend on duration of exposure (short-and long-term exposure). A significant association was found in healthy individuals as well as vulnerable populations.

Discussion
This systematic review assembled the currently available evidence on the impact of air pollution on human gut microbiota. All 10 revised studies evidenced that the exposure to air pollutants has the potential to alter the composition of gut microbiota [36][37][38][39][40][41][42][43][44][45]; some of them also correlated air pollution exposure to negative effects on human health via alterations in composition and/or function of the gut microbiota [36,38,41,43,45], and only three studies characterized some mechanisms by which air pollutants may have adverse health effects via the dysregulation of gut microbiota [37,39,42].
Concerning the effects of air pollution on the composition of gut microbiota, long-term (from 6 months up to 24 months) exposure to O 3 , NO 2 , SO 2 PM 10 , PM 2.5 , and PM 1 , as well as to traffic-related air-pollution (TRAP), has been shown to alter richness and diversity of human gut microbiota independently from age and gender of the study's populations. For example, higher O 3 exposure was associated with a higher abundance of bacterial species belonging to the Bacteroidaceae family [39]. Higher NO 2 levels were associated with a higher abundance of Coriobacteriaceae [39], and freeway TRAP exposure was correlated to decreased Bacteroidaceae and increased Coriobacteriaceae [45]. Lastly, short-time exposure to air pollutants (PM 2.5 for 3 days) was also demonstrated to induce alterations in gut microbiota, characterized by an increased abundance of a varied mix of both beneficial and harmful bacteria [37].
Several interesting evidence showed that the interplay between gut microbiota and air pollution also possessed negative effects on human health. Shifts in the relative abundance of Eggerthella, Phascolarctobacterium, and Clostridium have been described to partially mediate the effects of multiple air pollutants (PM 2.5 , PM 10 , O 3 , NO 2 , and SO 2 ) on adverse pregnancy outcomes [38]. In young adults, TRAP-mediated decreased abundances of Bacteroidaceae and increased abundances of Coriobacteriaceae were correlated with impaired fasting glucose, a known risk factor for type 2 diabetes [45]. Lastly, in adults, a decreased abundance of Firmicutes, Proteobacteria, and Verrucomicrobia, induced by PM 2.5 and PM 1 exposure, increased the risk of impaired fasting glucose and type 2 diabetes [36].
New insights into the mechanisms by which air pollutants can cause adverse health effects via the dysregulation of gut microbiota are of pathological importance. On this regard, functional microbial gene pathways related to cell growth and insulin release (Lornithine de novo biosynthesis, pantothenate, and coenzyme A biosynthesis I) were found to be associated with O 3 exposure. This supported the etiological role of gut microbiota in the association between air pollution and metabolic disorders, such as type 2 diabetes [39]. Zheng et al. suggested that the smog-dependant shift of gut microbiota with reduced abundances of Clostridia may trigger asthma attacks [41]. As a matter of fact, Clostridia can synthesize propionic acid or butyrates, which are known to stimulate the production of regulatory T cells that influence airway health indirectly through the gut-lung axis [46]. Interestingly, air pollution may simultaneously impact gut and lung health because, over the years, it has been proved that gut and lungs can communicate and influence each other, and they are connected via blood circulation and lymphatic system [47,48]. As a result, immune cells, cytokines, chemokines, and microbial metabolites can travel from an organ to the other, affecting its health. In this scenario, it is likely that lung inflammation and shifts in lung microbiota, following the exposure to air pollution, may lead to gut dysbiosis and, hence, to the onset or progression of intestinal pathologies.
Different potential mechanisms of damage were also described for other air pollutants; PM 2.5 may affect sphingolipid metabolism, which is partially mediated by the decreased abundance of Shuttleworthia, contributing to insulin resistance and, hence, type 2 diabetes [42]. Another study suggested that PM 2.5 exposure may result in changes in tryptophan metabolism, which is associated with the imbalance in the gut microbiota, thereby activating the gut-brain axis through the production of central neurotransmitters such as serotonin [36]. Specifically, tryptophan, a precursor of serotonin, is stored in limited quantities in the brain, and continuous supplementation from the digestive system is required for brain development and function [49].
The main strengths of the reviewed studies are the characterization of gut microbiota via high-throughput sequencing technologies (16S rDNA sequencing and shotgun metagenomics), as well as the accurate analysis of outdoor air pollutants. Moreover, the application of stringent inclusion criteria allowed greatly diminishing the impact of confounding bias related to the selection of the study population. In this regards, potentially important confounder factors known to be associated with the exposure to air pollutants and/or with the gut microbiota (for example, smoke or therapy with antibiotics) were considered.
Despite all the compelling evidence highlighted in this systematic review, a long road is still ahead of us before reaching clinically relevant conclusions due to the small number of papers (n = 10) and their weaknesses.
First, a high heterogeneity of sample size and type, as well as of exposure time, methods of exposure assessment, and metagenomic analysis, was observed. In particular, sample sizes were very variable among studies; notably, two studies [41,45] enrolled a low number of individuals (n = 21 and 42, respectively), and their results should be considered with caution. Moreover, the study populations included different categories of individuals, including pregnant women [38], children [41], young adults [39,45], adults [36,40,43], and older adults [37,42,44], with ages ranging from 5 to over 70 years. In this regard, it is well known that gut microbiota composition and diversity vary from infants to the elderly [50]. Of note, studies also included vulnerable populations, such as obese adolescents, asthmatic children, elderly, and pregnant women, which might be more susceptible to the negative effects of air pollution exposure. Concerning exposure time and assessment, these also varied among studies; time of exposure ranged from 3 days [37,42] to two years [36], which may lead to different changes in gut microbial composition. Daily air quality index scores assigned using the monitoring data of the China National Environmental Monitoring Centre [38], levels of urbanization [40], ambient monitoring station [41], realtime personal exposure [37,42], passive sampling devices [44], and other various modelled estimates [36,39,43,45] were used. It is well-known that the usage of different approaches in the assessment of air pollution exposure results in different accuracies and precision of estimates [51]. The most accurate method for assessing pollutant exposure seems to be personal monitoring, particularly biological monitoring; however, when the number of enrolled subjects is high, determining air pollutants levels can be suitable by using a variety of exposure assessment models [51]. This issue should be carefully considered for future research in the field. Lastly, it is very challenging to compare the biodiversity and the composition of the gut microbiota between different studies and patient groups due to different primers and hypervariable regions of 16S rDNA chosen for sequencing, alongside the numerous and diverse statistical measures used for bioinformatic analyses.
Second, almost all reviewed studies were observational, limiting the ability to provide a proof of causality.
Third, most of the examined studies enrolled mostly Chinese populations with distinct eating habits known to have impacts on the composition and diversity of the human gut microbiota [52]. The considerable disease burden attributable to air pollution following rapid economic development might explain the high number of studies in China [53].
Another relevant issue emerging from this review is that the overall risk of bias was partially considered in individual studies. For example, important information such as indoor activity time (home, school, and university) and indoor air pollution exposure, known to influence the composition of gut microbiota [54], was not considered. Moreover, the interplay between the different air pollutants and gut microbiota in the onset and development of chronic diseases has not been investigated. Similarly, nutrition and diet effects cannot be fully controlled, considering the long-term exposure to air pollution in most studies (>50%).

Conclusions
Overall, the exposure to air pollutants may be able to induce significant alterations in the composition of the gut microbiota. In addition, potential mechanisms by which air pollution may contribute to adverse health effects, such as impaired fasting glucose, adverse pregnancy outcomes, and asthma attacks via alterations in composition and/or function of the gut microbiota, were reported. However, the current evidence is weak due to the small number of papers, observational design of the included studies, as well as the high heterogeneity of the methods used for human microbiome and air pollution analyses. As a result, it will be of great interest to reach a consensus on the several parameters involved in designing metagenomic studies related to air pollution. Surely, an improved standardization of the methodological approach, from sample collection and storage to DNA extraction, genome amplification, and sequencing methods, is needed to obtain increased data accuracy, reproducibility, and comparability of results. In the future, large cohort studies worldwide based on simultaneous stool and respiratory sampling with the application of STORMS checklist for metagenomic analysis [35], as well as biological monitoring, will allow gaining deeper insights into the impact of air pollution on the growing prevalence of chronic diseases, although this study type is expensive and time consuming.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/toxics10100579/s1, Table S1: NOS score of the studies included in the systematic review.