The transient but not resident (TBNR) microbiome

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The transient but not resident (TBNR) microbiome: A Yin Yang model for lung immune system

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DOI: 10.3109/08958378.2015.1070220

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ISSN: 0895-8378 (print), 1091-7691 (electronic)

Inhal Toxicol, Early Online: 1–11

!2015 Taylor & Francis. DOI: 10.3109/08958378.2015.1070220

REVIEW ARTICLE

The transient but not resident (TBNR) microbiome: a Yin Yang model for lung immune system

Pardis Saeedi¹, Jafar Salimian², Ali Ahmadi¹, and Abbas Ali Imani Fooladi¹

1Applied Microbiology Research Center and²Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

Abstract

The concept of microbial content of the lung is still controversial. What make this more complicated are controversial results obtaining from different methodologies about lung microbiome and the definition of ‘‘lung sterility’’. Lungs may have very low bacteria but are not completely germ-free. Bacteria are constantly entering from the upper respiratory tract, but are then quickly being cleared. We can find bacterial DNA in the lungs, but it is much harder to ask about living bacteria. Here, we propose that if there is any trafficking of the microorganisms in the lung, it should be a ‘‘Transient But Not Resident (TBNR)’’ model. So, we speculate a

"Yin Yang model" for the lung immune system and TBNR. Despite beneficial roles of microbiome on the development of lung immune system, any disruption and alteration in the microbiota composition of upper and lower airways may trigger or lead to several diseases such as asthma, chronic obstructive pulmonary disease and mustard lung disease.

Keywords

Asthma, COPD, lung immune system, lung microbiome, lung sterility, mustard, TBNR microbiome

History

Received 27 January 2015 Revised 19 April 2015 Accepted 3 July 2015

Published online 24 August 2015

Introduction

The human body mucosal surfaces comprise a heterogeneous microbial composition which regulates the immune responses and immune homeostasis of the body. There is a close relationship between body health and body microbiota. In several diseases, immune functions of these mucosa may alter, which in turn, may cause various pathological complications. Although many researchers recently believe that the lower airway is not sterile, it seems that the lung is not essentially harbor resident organisms. From upper airways, the organisms may constantly enter downward to the lung and then be eliminated by the lung defense systems. Here, we speculate that unlike other mucosal surfaces, the lung has a Transient But Not Resident (TBNR) microbiome pattern, which establish a dynamic relation with the immune system.

In this review, we try to define microbiome in general, and the lung microbiome and its origin, in particular. Also, we compare the germ-free and conventional animals in terms of their immune structural and functional differences due to the presence of lung microbiome. Then, we study the role of microbiome in the development and homeostasis of lung immune system, and interaction between lung immune responses and lung microbiome (Yin Yang model). Finally, we describe the factors altering the content of the lung

microbiome, which can lead to some diseases such as asthma and allergy, chronic obstructive pulmonary disease (COPD) and mustard lung disease.

The microbiome: definition

The ‘‘microbiome’’ is the total collection of viable and non- viable microorganisms and their products on the skin or mucosal surfaces of the human body [e.g. gastrointestinal (GI), urogenital and respiratory tracts] (Collado et al., 2012;

Grice & Segre, 2012; Ubeda & Pamer, 2012; Ursell et al., 2012). Two other related terms are ‘‘microbiota’’, that are all viable microbial community and taxa existing in humans (Grice & Segre, 2012; Petersen & Round, 2014); and

‘‘microflora’’, that are microorganisms that inhabit certain areas of the body (a subset of microbiota). There are about 10¹⁴symbiotic microbial cells, tenfold more than total human body cells (10¹³), which make up around 2% of total body mass and consist of commensal bacteria and fungi (Cho &

Blaser, 2012; Collado et al., 2012; Garzoni et al., 2013; Sze et al., 2014; Turnbaugh et al., 2007; Ursell et al., 2012; Yi &

Li, 2012). The concept of human microbiome, the collection of all human microorganisms and their products (Collado et al., 2012; Petersen & Round, 2014), was unraveled by evolving several methods (such as DNA sequencing, germ free-animal models, diversity profiling, the operational taxo- nomic units, and comparing core and transient microbiota) (Ursell et al., 2012). Besides being critical for organ development and health condition, the human microbiome and its components contribute in developing and even differentiating the host immune system. Microbiota composition may have a variation in number and diversity among Address for correspondence: Abbas Ali Imani Fooladi, Applied

Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. E-mail: [email protected] and imani- [email protected]

Jafar Salimian, Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. E-mail: jafar.salimian@

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different individuals, which in turn, may change during their lifespan. In spite of all crucial and beneficial roles, any disruption in the microbiota composition (i.e. dysbiosis or microbial imbalance) may trigger or lead to several diseases (Littman & Pamer, 2011; Ubeda & Pamer, 2012).

The lung microbiome

Still remain controversial issue?

The airways microbiome is the entire community of microorganisms and their components that inhabit the respiratory tract (Garzoni et al., 2013; Gollwitzer & Marsland, 2014; Sze et al., 2014). The upper respiratory tract (URT) (nasopharynx, oropharynx and laryngopharynx) is colonized with a vast number of anaerobes and tenfold more aerobic bacteria. In the lower airways, the most common anaerobic bacteria found are Peptostreptococcus, Veillonella, Actinomyces and Fusobac- terium species, and the furthermost typical aerobic bacteria include Streptococcus, Haemophilus and Neisseria species (Murray et al., 2013); mostly influenced by the individual’s environment, gut microbiome and nutrition (Gollwitzer &

Marsland, 2014; Madan et al., 2012). The presence of normal flora in lower respiratory tract (LRT) (larynx, trachea, bronchioles and lower airways) is a controversial issue. At first, it is worth mentioning that there are no universal and validated criteria for defining the concept of ‘‘sterility’’ and especially ‘‘sterility in lungs’’. Whether the sterility is defined as lack of living microorganisms (undetectable in pure cultures), or the presence of rare transient bacteria and/or any microbial components such as nucleic acids and structural/secretory products are the questions to be addressed. In fact, it all depends on what we mean by sterility. Lungs may have very low bacteria but are not completely germ-free.

Bacteria are constantly entering from the URT, but are then being cleared. We can find bacterial DNA in the lungs, but it is much harder to ask about living bacteria.

Taken together, we propose that if there is any trafficking of the microorganisms in the lung, it should be a transient model, but not resident microbiota. As a result, there is TBNR microbiome in the bronchial tree and lung. The key concept is the time of presence of organisms in that location; whether the bacteria have short-term visa for inhabiting there, or according to Zakharkina et al. (2013), there is a colonization and long validity visa for bacterial passengers to reside in the lung?

Early studies applying traditional methods indicated that the lower airway in healthy persons is sterile; but modern analytical techniques, such as 16S rRNA sequencing, disclose a new concept supporting the microbial presence in the human lower airways. As pioneers, Laurenzi et al. (1961), Pecora (1963), Austrian (1968) and also Murray et al. (2013), based on traditional culture-based methods, have revealed that

‘‘the lungs are sterile organs’’ (Austrian, 1968; Laurenzi et al., 1961; Murray et al., 2013; Pecora, 1963). On the other hand, other studies by Monso et al. (1999), Harris et al.

(2007), Bousbia et al. (2010), Hilty et al. (2010), Charlson et al. (2011), Erb-Downward et al. (2011) and Huang et al.

(2011), Sze et al. (2012), Duff et al. (2013), Garzoni et al.

(2013), Rogers et al. (2013) and Segal et al. (2013) and Sze et al. (2014) supported the contradictory concept that lungs

are not sterile using culture-independent methods based on genomics and metagenomics (Bousbia et al., 2010; Charlson et al., 2011; Duff et al., 2013; Erb-Downward et al., 2011;

Garzoni et al., 2013; Harris et al., 2007; Hilty et al., 2010, Huang et al., 2011; Monso et al., 1999; Rogers et al., 2013;

Segal et al., 2013; Sze et al., 2012, 2014). Thus, as listed in Table 1, different studies have resulted in distinct outcomes for and against the presence of lung microbiome. Herein, some points are considerable. First, the culture-dependent methods have lower detection limits than culture-independent methods (e.g. 16S rRNA sequence analysis and quanitative PCR). Second, the specimen type is critical. For example, nasopharyngial and oropharyngial swabs are not as valuable as bronchoalveolar lavage fluid (BALF) and brushing samples, mainly due to their cross contamination. Third, many lower airway bacteria are fastidious and need selective, enriched culture media and an experienced technician. Fourth, VBNC (viable but not culturable) bacteria may exist as microflora which cannot recover by traditional culturing.

Fifth, the culture-independent methods have been established on detection of microbial nucleic acids, instead of detecting living bacteria in the lower airways. Moreover, it is note worthy that the volume of sample and statistical analyses are essential to a valid interpretation. Furthermore, a few bacteria present in LRT may be detectable by molecular but not cultural methods, due to the fact that these organisms should not be considered as definite LRT microbiota. It seems that they could originate from microaspiration from the URT (Berger & Wunderink, 2013; Charlson et al., 2011, 2012a;

Pragman et al., 2012; Segal et al., 2013; Sze et al., 2014;

Zakharkina et al., 2013) or may come down through bronchoscopic contamination (Charlson et al., 2011, 2012b;

Lozupone et al., 2013; Pragman et al., 2012; Zakharkina et al., 2013). Conflicting conclusions about the lung microbiome among different studies is most likely due to the methodological differences, sample collection techniques, specimen volume and type, population variances, topography and alterations in DNA sequencing approaches, and the data analyses (Morris et al., 2013).

To sum up, we conclude that the LRT is not sterile since the lungs are constantly exposed to environmental microbes and those from the upper airways, so may not be considered to be completely free of bacteria.

Origin of lung microbiome

The hypothesis supporting the non-sterility of LRT poses a new question about the primary source of the lower airways microbiome (Garzoni et al., 2013; Gollwitzer & Marsland, 2014). There are some possible explanations including the impact of the environment, gut microbiome, composition of the lower airways microbiota (Madan et al., 2012), and even URT cross contamination via sampling (Gollwitzer &

Marsland, 2014).

It is obvious that the origin of major population of lower airway microbiome is from the upper airways. Moreover, there is small proportion of bacteria that are capable of tolerating the unique condition of the lungs (Shaikh-Lesko, 2014). Nevertheless, there are few reports using modern molecular techniques indicating the presence of distinctive Inhalation Toxicology Downloaded from informahealthcare.com by 188.34.160.243 on 08/24/15 For personal use only.

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and persistent bacteria in the lower airways (Gollwitzer &

Marsland, 2014; Sze et al., 2012). Table 2 illustrates that the lower airways harbor similar and exclusive microbiota. Those that are similar to what is found in the upper airways descending to the lungs, but those that are not similar may be environmental microorganisms able to survive by the absence of sufficient microbial load in the lower airways competing with them.

Evidence for presence of lung microbiome:

comparison between germ free and conventional animals

The role of microbiome in immune system development is supported by comparing the conventional and germ-free animals. Germ-free animals anatomically and physiologically have underdeveloped immune system. For example, they have fewer and smaller lymph nodes, fewer plasma cells and lymphocytes, lower Ig levels (especially IgA), lower TLR (Toll Like Receptor) expression and more susceptibility to pathogens (Round & Mazmanian, 2009). In mucosal sites (airway, GI and urogenital tracts), the lack of microbial colonization can cause partial development of mucosa associated lymphoid tissue, and subsequently decreasing B and T lymphocytes and sIgA, decreased production of

particular antimicrobial proteins (e.g. defensin) and mucus accumulation due to mucociliary defects (Round &

Mazmanian, 2009). In these animals, upon re-exposure to foreign microbes, mucosal immunity started to develop (Gaboriau-Routhiau et al., 2009; Mazmanian et al., 2005;

Salzman, 2011). Table 3 summarizes some studies comparing immune system components between germ-free and conventional animals.

Lung immune system and microbiome Airways immune system structure

Human airways are exposed to a large number of pollutants, infectious pathogens and allergens. So, in order to establish a normal and effective immune response, some specific local tissues are needed in airways. Nasal associated lymphoid tissue (NALT) and bronchus-associated lymphoid tissue (BALT) are widely diffused lymphoid tissues present throughout the airways. In addition, highly organized and specialized mucosal lymphoid tissues (palatine, tubal and lingual tonsils, and adenoids, so called the Waldeyer’s ring) are present in the NALT and the BALT is widely diffused lymphoid tissues lining the airways. Furthermore, other non- lymphoid areas such as the bronchial-alveolar parenchyma can promote immune reactions and thus establish different Table 1. Studies with different methodologies that is indicative of the presence or absence of lungs microbiota.

Methodology Sampling Result References

1. Culture-based method (The LRT is sterile^a)

2485 Sputum/Nasophrynx swabs

Identification of upper microbiome

Austrian (1968) Culture of BALF/swab/44

patients

Identification of lung pathogens

Pecora (1963) Pharynx swab/Sputum/

BALF/52 patients

Identification of lung pathogens

Laurenzi et al. (1961) 2. Culture-independent

methods (the LRT is not sterile)

2.1. 16S rRNA sequence analysis

Naso/Oropharyngial swabs/

BALF/6 healthy control cases

There was no unique lung microbiome

Charlson et al. (2011, 2012b)

BALF/10 healthy control cases

The composition of the lung microbiota was determined

Pragman et al. (2012)

Swabs/brushings/BALF/8 healthy control cases

The bronchial tree contains a characteristic microbiota

Hilty et al. (2010) Swabs/BALF/9 healthy

control cases

Healthy lungs harbor microbiome

Garzoni et al. (2013) BALF/7 healthy smokers

and 3 none smokers

Similar aerobic and anaerobic genera have been detected, using culture/

molecular methods

Erb-Downward et al. (2011)

BALF/oral wash/64 healthy cases

Many specific bacteria in the lungs

Morris et al. (2013) 2.2. 16S rRNA sequence

analysis and qPCR

BALF/210 patients in ICU Pathogen strain in 6 cases Bousbia et al. (2010) BALF/29 asymptomatics

cases

Similar microbiome to UTR

Segal et al. (2013) BALF/77 HIV-negative

cases

Pathogen strain in one case Lozupone et al. (2013) 2.3. 16S rRNA sequence

analysis and qPCR and TRFLP

Surgical lung tissue samples/8 healthy non- smoker cases

Specific bacteria in the lungs

Sze et al. (2012)

BALF/14 control cases Molecular identification of bacteria

Harris et al. (2007) BALF/9 healthy control

cases

A diverse microbiome in healthy cases

Zakharkina et al. (2013)

LRT, lower respiratory tract; UTR, upper respiratory tract; BALF, bronchoalveolar lavage fluid; TRFLP, terminal-restriction fragment length polymorphism.

aSterile:lack of viable microorganism.

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types of immune tissues in the host (Williams, 2011).

The lung innate immune system contains the mucus, mucocilliary apparatus, serum products, epithelia, dendritic cells and other types of leukocytes (Derscheid & Ackermann, 2013) (Figure 1).

Role of microbiome in development and homeostasis of lung immune system

The human immune system is divided into innate and adaptive immune responses. In the innate immune system, pattern recognition receptors [PRRs, e.g. TLR, NLR Table 3. Comparison of germ-free (GF) and conventional (CV) animals’ immune system components.

Cases GF versus CV animals References

Airways immune system anatomy

Lymph nodes Fewer and smaller Round & Mazmanian (2009) and Yi & Li (2012)

Airways immune system function

Airway hyper responsiveness (AHR) Increased Herbst et al. (2011)

Serum immunoglobin levels Lower Round & Mazmanian (2009)

Production of secretory IgA Reduced Herbst et al. (2011), Round & Mazmanian

(2009), Vieira et al. (1998), Yi & Li (2012)

Production of IgE Increased Herbst et al. (2011) and Hazebrouck et al. (2009)

Th1 responses Less Herbst et al. (2011), Gollwitzer & Marsland

(2014), Gollwitzer et al. (2014)

Th2 responses More Herbst et al. (2011), Gollwitzer & Marsland

(2014) and Gollwitzer et al. (2014)

Treg cells Lower and not effective Hazebrouck et al. (2009), Littman & Pamer

(2011), Ostman et al. (2006) and Round &

Mazmanian (2009) Immune cells levels

Levels of leukocytes Lower Round & Mazmanian (2009)

Alveolar macrophages and dendritic cells Decreased Herbst et al. (2011)

Antigen-presenting cells more efficient Gollwitzer & Marsland (2014)

NK cells in mucosa Much more Ostman et al. (2006)

NK cell priming and antiviral immunity severely compromised Thaiss et al. (2014) Basophils (producers of IL4¼allergic response) Increased Herbst et al. (2011)

Th17 cells Absent Littman & Pamer (2011)

CD4 + T cells Fewer Littman & Pamer (2011) and Round &

Mazmanian (2009)

CD8 + T cells Reduced cytotoxicity Round & Mazmanian (2009)

Cell products

Expression of MHC class II molecules Reduced Round & Mazmanian (2009)

Expression and localization of PRRs (TLRs) Reduced and defective Round & Mazmanian (2009) and Sharma et al.

(2012)

Levels of IL25 Reduced Round & Mazmanian (2009)

IL17 Decreased Littman & Pamer (2011) and Prabhudas et al.

(2011)

Induce expression of various IFN Failed Thaiss et al. (2014)

IL6, TNFa, IL12, IL1 and IL18 Failed Thaiss et al. (2014) and Vieira et al. (1998)

Expression of AMPs Defective Round & Mazmanian (2009)

IL12 and early IFNgproduction The same Vieira et al. (1998)

Cells producing IL10 The same Herbst et al. (2011)

Production of IL10 and IL13 by splenocytes Reduced Ostman et al. (2006)

Table 2. The profile of microbiota existed in lower airways which can be similar to upper airways or unique.

Lower airways harbor Bacteria References

Similar microbiota to upper Moraxellaceae, Comamonadaceae, Aerococcaceae, Rhizobiaceae, Lactobacillaceae, Nocardiaceae, Capnocytophagaceae, Microbacteriaceae, Sphingomonadaceae, Flavobacteriaceae, Peptostreptococcaceae, Fusobacteriaceae, Staphylococcaceae, Porphyromonadaceae, Pasteurellaceae, Neisseriaceae, Acidaminococcaceae, Streptococcaceae, Prevotellaceae

Garzoni et al. (2013)

Pseudomonas, Streptococcus, PrevotellaandFusobacterium, Haemophilus, Veillonella,andPorphyromonas

Erb-Downward et al. (2011) Corynebacterium, Prevotella, Staphylococcus, Streptococcus, Veilonella,

HaemophilusandNeisseria

Hilty et al. (2010)

Unique bacteria Tropherymawhipplei Berger & Wunderink (2013)

and Charlson et al. (2011) Comamonadaceae, Diaphorobacter unclassified, Brevundimonasdiminuta Sze et al. (2012)

Prevotella, Porphyromonas, leptotrichia, Neisseria elcogata, Acinetobacterjohnsoni, Haemophilus influanzae

Hilty et al. (2010)

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(NOD-like receptors)] are scanning microbial components.

After binding of ligands to their related TLRs, signaling pathways through adaptor proteins [e.g. MyD88 or TRIF (TIR-domain-containing adapter-inducing interferon-b)]

induced various gene expressions in innate immune cells (e.g. dendritic cells) (Chang, 2012; Sharma et al., 2012).

These genes include inflammatory or regulatory cytokines, phagocytosis mediators, co-stimulatory and other molecules involved in antigen presentation. T cells recognize antigens in MHC cleft and become activated via cytokine and co- stimulatory molecules and differentiate into helper cells such as Th1 (cell mediated immunity), Th2 (humoral immunity), Th17 (inflammatory condition) or Treg (regulatory T cell) (Han et al., 2012; Ivanov et al., 2009).

Development of human immune system startsin uteroand continues postnatal. Before birth, the default pattern of immune system is dictated by Th2 responses. Following birth, Th2 polarity in the fetal immune system will switch to a non-allergic Th1 phenotype (Azad & Kozyrskyj, 2012; Holt et al., 2005; Leme et al., 2006). In fact, after birth, inhalation of air microorganisms can cause immune system development in the neonatal lung. In a normal state, inhalation of air microorganisms promote TLR expression (especially TLR9 in the lung) and direct immune system response toward cell- mediated immunity (Th1 cells) and production of IFNg and TNFa in the lung. It seems that air microorganisms or their products bind to PRRs and can stimulate dendritic cells and shift naı¨ve T cells to Th1 cells (Imani Fooladi et al., 2011). In fact, the fetus produces high levels of anti-inflammatory cytokines like IL10 and TGFb(Sharma et al., 2012), but has very low levels of pro-inflammatory cytokines such as IL1b, IL6, TNFaand IFNa(Strunk et al., 2004). From infancy to adolescence, the production of lung Th1 cytokines such as IFNg, IL12 and IFNa is increased (Figure 2). This phenomenon has a crucial role to prevent lung immune response shift

into the Th2 pattern. Principally, it is an inverse relationship between IFNg level and serum IgE. Immune system development into Th1 protects against neonatal asthma and allergic disease. Non-existence of microbiome in germ free mice can cause immune response development toward Th2 type and leads to allergic reaction. In a mouse model, mucosal administration of component or whole bacterium of innocuous Escherichia coli such as lipopeptide (stimulator of Figure 1. The schematic structure of lung innate immune system. The lung innate immune system contains epithelial cells and the mucus, mucocilliary apparatus, serum products, epithelial cell, dendritic cells and other types of leukocytes. TLR, toll like receptor; AMP, antimicrobial peptides; DC, dendretic cell; MQ, macrophage; E, eosinophil; B, B cell; T, T cell.

Figure 2. Development of lung immune system before and after birth.

Before birth, default of lung immune system is Th2 type. After birth, neonatal airways were colonized by air microorganisms (TBNR) that trigger TLR expression. This TBNR microbiome induces lung immune system and shift naı¨ve T cells toward Th1 Type. In pathologic condition, alteration in microbiota can change immune system response into Th2 or Th17 types (Renz et al., 2012).

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TLR2), peptidoglycan (TLR2, TLR4), LPS (TLR4) and DNA (TLR9) can induce Th1 immune response and protect mice against asthma and allergy (Marsland et al., 2013; Thaiss et al., 2014). In most cases, the absence of Th2 response means that immune response has switched to Th1 type. Also, nasal administration of innocuous Gram-positive and Gram- negative bacteria induces Th1 type immune response (Gollwitzer et al., 2014). Several studies have used probiotics (oral administration) as a safe and noninvasive strategy to ameliorate some diseases. Through balancing the resident microbiota, it regulates immune responses in mucosal surfaces, moderates pathophysiological symptoms and diminishes the prevalence of allergy and cancers (Guandalini et al., 2015; Imani Fooladi et al., 2013; Khani et al., 2012).

Apart from airways microbiome, the non-airway microbiome also has an important role in the lung immune- regulation (Th1/Th2 balancing). GI microbiome induces regulatory T cells that help keep the balance of Th1/Th2. In addition, recognition of microbiota compositions by PRRs enhances systemic innate immunity (Azad & Kozyrskyj, 2012; Kaplan et al., 2011). For maintaining the homeostasis of intestinal cells, PRRs sense microbial compounds which induce Treg and Th17 cells (Ichinohe et al., 2011). The most significant functional role of the human microbiota is mainly in the GI tract and can be involved in the maturation of the host immune system. Despite beneficial roles, such as critical production of microelements and assistance in metabolism, disruptions in the microbiota may cause some diseases (Garzoni et al., 2013). The GI microbiome has a profound effect on shaping and regulating the whole host immune system as well as pulmonary immune responses by: (1) activating the gut dendritic cells and regulating the systemic balance of Th17 cells and Treg cells (Atarashi et al., 2011;

Han et al., 2012; Ivanov et al., 2008, 2009; Ostman et al., 2006; Worbs et al., 2006); (2) shaping the systemic normal tolerance (Abraham & Cho, 2009; Han et al., 2012); (3) mucosal maturation and immune response establishment by evolving gut-associated lymphoid tissues including Peyer’s patches, isolated lymphoid follicles and lymph nodes, (Chervonsky, 2009; Hooper & Gordon, 2001) which are underdeveloped in early infants and germ-free animals (Ichinohe et al., 2011; Round & Mazmanian, 2009).

The role of airway microbiome in diseases

As mentioned above, microbiome has an important role in physiological homeostasis of the body. It most likely acts through colonization competition, maintenance of epithelial integrity and immune system regulation (Erb-Downward et al., 2011). Although the precise effect of airway microbiome in diseases is a complicated issue (Han et al., 2012;

Huang et al., 2013; Huang & Lynch, 2011), many researchers have demonstrated that the community of airway microbiome may change between healthy and diseased conditions (Huang

& Lynch, 2011; Zakharkina et al., 2013). There are some factors that seem to contribute to alterations (stated below) in airway microbiota that, in turn, may lead to various subsequent infectious and non-infectious diseases. Here we sum- marize these items in more detail.

Factors altering the content of lung microbiota

There are some factors which disrupt the lower airway microbiota and may lead to several chronic lung diseases (Erb-Downward et al., 2011; Garzoni et al., 2013; Han et al., 2012; Hilty et al., 2010; Pragman et al., 2012; Sze et al., 2012). These factors may be classified as: (I) Anatomical injuries (either inherited or acquired) including genetic malfunctions, mucociliary dysfunction, traumas, surgeries and burns; (II) Pathological effects including microbial infections, prolonged antibiotic therapy and exposure to hazardous chemicals; (III) Physiological changes such as hormonal alterations (e.g. puberty, menopause, nutrition and stress) and finally, (IV) Immune system defects including defects in B and T cells and phagocytes, antibodies and complement system. Bacterial colonization in the LRT may be a consequence of immune system impairment. These patients are not completely able to phagocytose the inhaled particles and bacteria and also deficient in efferocytosis of apoptotic airway epithelial cells. Some researchers have declared that, phagocytosis requires adequate function of microtubules which has failed in COPD (Barnes, 2014;

Donnelly & Barnes, 2012; Hodge et al., 2003; Mu¨llerova et al., 2012; Taylor et al., 2010). Persistent inflammation and exacerbation in COPD patients is due to failure of phagocytes in clearance of bacterial pathogens and necrotic material elimination from the lung (Barnes, 2014; Donnelly & Barnes, 2012).

Emerging alterations in the lung microbiota

With respect to the type and degree of factors mentioned previously, the possible shifts in microbiome may include: (I) Reduction in microbiome complexity (as loss of diversity in bacterial composition in COPD (Erb-Downward et al., 2011;

Sze et al., 2014) and Crohn’s disease (Cho & Blaser, 2012);

(II) Specific microbiota abundance (e.g. due to antibiotic or antiretroviral therapy) (Lozupone et al., 2013) and (III) Increased microbial pathogenicity (due to acquiring toxin, epithelial adhesive molecules, and antibiotic resistant genes) through different genetic exchanges between diverse species and genera of the microbiota resulting in new genetic traits such as antibiotic resistance and virulence in human microbiome (Cho & Blaser, 2012; Liu et al., 2012; Smillie et al., 2011).

Diseases affected by microbiome disruption

Studies have made clear that disruption in the content of microbiome may lead to clinical outcomes. Now, the question is how microbiome can cause these clinical outcomes? Many efforts have been made to determine the causative relation between abundance and diversity of microbiome and pathological conditions (Cho & Blaser, 2012). Reduction of microbiome diversity and complexity is accompanied by the overrepresentation of particular microbial species which results in inflammation (Boyton et al., 2013; Han et al., 2012). Loss of keystone microbial species may trigger some cascade events (targeting the imbalance of immune regulation and increase of immune responses) that may lead to more tissue injuries (Cho & Blaser, 2012) (Figure 3). So, we Inhalation Toxicology Downloaded from informahealthcare.com by 188.34.160.243 on 08/24/15 For personal use only.

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conclude that there is a dynamic balance between the lung microbiome and immune system, as a ‘‘Yin and Yang phenomenon’’. Here are some diseases in which the role of microbiota and shifts of their composition are decisive.

Asthma and allergy

Some studies propose that the lower airways of patients with stable asthma may be colonized by distinctive microbiota and the microbiota community and diversity lead to bronchial hyper responsiveness (Huang & Lynch, 2011). There is some evidence that overprescribing or inappropriate use of antimicrobial agents in childhood may result in asthma and allergy development (Risnes et al., 2010), which is probably due to disruption of the GI microbiome, replacement of pathogens and perturbation of mechanisms of mucosal tolerance (Dickson et al., 2013; Huffnagle, 2010). A clear outgrowth in the proportion of Firmicutes (Streptococcus), Proteobacteria (particularly Haemophilus, Moraxella and Neisseria species), Pseudomonodaceae, Comamonadaceae, Oxalobacteriaceae, Shingomonadaceae in the LRT have been shown in asthmatic groups as compared with healthy controls (Dickson et al., 2013; Hilty et al., 2010; Marsland et al., 2013) which can be both, cause and effect of the disease. By contrast, there may be a reduction of Bacteroidetes and Prevotellaspecies which is a direct inhibitory effect of other bacteria (Beck et al., 2012; Hilty et al., 2010; Huang & Lynch, 2011; Huang et al., 2011). In both cases (increase or decrese of specific bacteria) dysbiosis or imbalance of microbiome leads to these diseases.

Chronic obstructive pulmonary disease

There is a correlation between COPD, the airflow obstruction subsequent to emphysema or chronic bronchitis, and the lower airway microbiome (Beck et al., 2012). Once microbiome imbalance happens, the bacterial phyla ofProteobacteria(in particularHaemophilusspp.), Firmicutes, Comamonadaceae, Shingomonadaceae, Oxalobacteriaceae, Pseudomonodaceae are overrepresented in the lungs of allergic and COPD patients, whereas Bacteroidetes and Prevotella are reduced (Beck et al., 2012; Dickson et al., 2013; Hilty et al., 2010;

Huang & Lynch, 2011; Huang et al., 2011; Marsland et al., 2013).

COPD patients are subjected to several other conditions resulting in acute worsening of superimposed chronic disease.

Despite the destructive impacts on the lungs, COPD should be considered as a complex systemic disease (Karadag et al., 2008). COPD exacerbation, a very common distressing event affecting some patients with moderate-to-severe COPD, impacts impressively on health state and alleviation of symptoms (Bourbeau, 2009). Apart from the clinical diversity of COPD, the nature and course of inflammation commonly affects the airways, which exacerbates with the progress of disease. There is evidence of the impact of microbiota alteration on triggering inflammatory events. The lungs inflammatory response initiates by infiltration of inflammatory and immune cells leading to tissue destruction, alveolar airspace enlargement and disease progression (Stefanska &

Walsh, 2009; Sze et al., 2014). There is also evidence for the existence of auto-immunity contributing to COPD Figure 3. Illustration of the lung microbiome disruption resulting in vicious cycle.

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progression. The presentation of endogenous antigens com- mences tissue destruction and gives rise to a classical autoimmune response (Stefanska & Walsh, 2009).

Mustard lung disease

Sulfur mustard (or mustard gas, SM [2,2⁰-dichlorodiethyl sulfide]) is a potent chemical vesicant (blistering) with extensive morbidity (Ghasemi et al., 2009; Kehe & Szinicz, 2005; Malaviya et al., 2010; Roshan et al., 2013; Shams et al., 2009). It is a toxic DNA alkylating agent and identified as a human carcinogen (Jafari & Ghanei, 2010; Lari et al., 2012;

Roshan et al., 2013; Shams et al., 2009; Steinritz et al., 2007).

It was used during the World War I and in the Iraq-Iran war during 1980–1988 (Balali-Mood & Hefazi, 2006). Over 100 000 people were subjected to severe injuries and approximately 45 000 patients continue suffering from long term exposure (Roshan et al., 2013). SM has both short- and long-term toxic effects in the eyes, skin, nervous system, respiratory and hematopoietic systems (Balali-Mood &

Hefazi, 2006; Ghasemi et al., 2013, Ghazanfari et al., 2009, 2013; Lari et al., 2014; Shohrati et al., 2007; Yaraee et al., 2009). The respiratory epithelial cells are very susceptible to the effects of SM exposure and undergo sloughing which enhance airways secretions and nasal discharge, then airway obstruction, and even bronchospasm. After acute mustard exposure, airway damage leads to pulmonary insufficiency and infection (Razavi et al., 2013). Hematological results indicated a major drop in WBC and an increase in lymphocytes (Ayyobi et al., 2014; Ghazanfari et al., 2013; Mirzamani et al., 2012), as well as neutrophilia (Beheshti et al., 2006;

Ebrahimi et al., 2010).

According to some Iranian researchers’ findings in individuals chronically exposed to SM, there is elevation in expression levels of TGF-b1, TGF-b2, C-fos, NF-kB and inflammatory cytokines (Pourfarzam et al., 2009), but reduction in heme oxygenase in airways epithelial cells (Imanifooladi et al., 2010; Nourani et al., 2010, 2011), and efferocytosis (Pirzad et al., 2011; Zarin et al., 2010). The serum level of some chemokines such as SDF-1a (Stromal- Derived Factor 1a or CXCL12) is reduced (Ayyobi et al., 2014). Mitigating vesicant effect and toxicity of SM requires operative strategies. Inhaled corticosteroids, long-acting b2- agonists and macrolide antibiotics are effective in treatment of these patients (Ghanei et al., 2007). However, steroid treatment can disrupt the microbial balance.

SM can directly affect or eliminate some lower airway microbiome by producing free radicals and sloughing (Brimfield et al., 2012; Graham et al., 2005; Sadraei et al., 2011). On the other hand, mucuciliary clearance deficiency helps microbial pathogen colonization to lower airways of mustard-exposed lungs. Inspecting microbial communities, in 25% of BAL samples, bacteria was observed including S. aureus, coagulase-negative Staphylococcus species and E. coli (Moghadam et al., 2003). Ghanei et al. reported the prevalence of bacteria in BAL culture of 79 lung mustard patients for the first time. It consists of 55% SNGA (non- group A streptococci), 13% Pseudomonas aeroginosa, 9%

enterobacter, 7%S. aureus, 6% SGA (group A streptococci) and 5%Klebsiellaand the other microorganisms totalling 5%.

AlsoH. influenza,S. pneumoniae andMoraxella catarrhalis are responsible for 85–95% of exacerbations. Besides, S.

viridansis found as pneumonia agent in COPDs.H. influenza is an intracellular pathogen, which accelerates the risk of chlamydia pnemoniaeinfections (Panahi et al., 2004).

In an experiment, Imani et al. utilized siRNA to inhibit opportunistic bacteria such as pseudomonas in veterans’

airways. They found that this treatment method was not efficient because of high growth speed of bacteria (Fooladi et al., 2013).

Conclusion

Despite the recent studies implying the presence of lung microbiome, the topic remains controversial. The final decision about the presence of lung microbiome may depend on the obtaining of completely sterile specimens, isolation of pure and live organisms (through culture based techniques) and precise characterization of isolated strains. It seems that some key questions should be addressed. Are the organisms resident? Or they are transient in the location (short validity visa). Can we apply the concept of TBNR microbiome or not? Is there any colonization? On the other hand, we cannot ignore the results of studies reporting about 2200 different genes in the bronchial tree in addition to the role of lower airway microbiome in the development and homeostasis of lung immune system between normal and germ-free animals. There is no doubt about the role of lung microbiome on the homeostasis of lung immune system. In fact, what we usually call ‘‘Yin-Yang phenomenon’’, is a dynamic balance between TBNR microbiome of lower airways and immune responses.

In conclusion, if considering microbiota as transient bacteria rather than resident (TBNR), prescribing steroids and antibiotics to ameliorate the lung inflammation can weaken immune system and favor the growth of opportunistic microorganisms. These therapeutic strategies cannot specif- ically be helpful in the treatment of mustard-exposed lungs.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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