Chronic rhinosinusitis (CRS) is an important public health problem and has a major impact on the quality of life [1–4]. The studies of CRS have been limited by access to tissue, the complexity of the sinonasal physiology, a lack of available biomarkers, the absence of useful animal models, a paucity of cohorts with biological samples for analysis, and limited well designed clinical trials or investigations of the immune function. Therefore, novel strategies for identifying biological mechanisms underlying this disease are much needed.
A number of studies have shown abnormalities in immune responses in CRS , and recent studies have highlighted the close relationship between the epithelium and immune system. Indeed, Th1, Th2, Th17, T regulatory (Treg) cells, neutrophils, and eosinophils as well as their associated cytokines and mediators have all been implicated in the resultant cellular and molecular immunopathology [6–13]. Moreover, common to these studies is the conclusion that the dysregulation of immune responses contributes to the striking inflammation seen . Factors that affect these responses may be involved in the persistent inflammation in CRS.
If we could understand what stimuli generate the hyperinflammatory responses in the sinonasal mucosa, we could better understand what factors affect this process. The aforementioned work has been performed under the prevailing framework of CRS as an inflammatory disease. Early efforts toward identifying the genesis of this inflammatory response initially focused on bacteria using culture techniques to assess the presence of organisms. Although studies show that some patients yield positive cultures from sinus samples, it has been difficult to generate support for this idea because a high proportion of CRS cultures fail to grow any organisms or yield bacteria with no obvious relationship to disease. The variability in recovery may result from differences in the methodologies used for collection, processing, transportation and cultivation, patient heterogeneity, geographical characteristics, and prior therapy, but also reflects the fact that many organisms do not grow in culture.
One must consider that culture-based methods of assessing the presence of bacteria fail to recognize a large set of organisms that are present in the human body. For example, gut cultivation-based technologies limit analysis because more than 80% of the estimated species are not readily cultivated . The cultures also fail to provide information on the communal composition and structure, phenotype, function, and gene expression of these organisms in their natural habitats. Therefore, we cannot discount the hypothesis that organisms from the sinuses that do not grow in culture influence the inflammation in CRS, either directly or indirectly, through effects on other organisms involved in disease. Similarly, environmental factors, such as smoking may affect the composition of these bacteria.
Studies on the total genomes of bacteria inhabiting the body, termed the microbiome, have recently been performed at National Institutes of Health. To elucidate the normal human microbiome, samples were collected from 242 healthy Americans (129 men and 113 women) [15▪▪]. Tissue samples were collected from 15 and 18 sites of the body from men and women, respectively. A maximum of three samples were collected from the oral and nasal cavities, skin (posterior regions of the bilateral ears and medial sides of the bilateral elbows), lower intestine (feces), and female vagina. Total microbial DNA was purified from each sample and analyzed using a DNA base sequencer. To identify the bacteria-specific gene (variable ribosomal RNA gene termed 16S rRNA), 3.5 trillion base pairs of genomic sequence data were arranged. Only bacterial DNA base sequences could be analyzed without analyzing human genomic DNA sequences. In addition, metabolic activities encoded by these microbial genes could be analyzed by metagenomic sequencing (or determination of the total DNA base sequences of the microorganisms). This Human Microbiome Project (HMP) demonstrated the presence of more than 10 000 microbial species in the human body, and it was estimated that 81–99% of the total microbial species inhabiting healthy individuals were identified.
Analysis of the microbiome in various diseases has also been progressing. For example, on analysis of the microbiome in the nasal cavity of children with fever of unknown origin, which frequently occurs in children, a maximum of five times larger amount of viral DNA was detected in nasal cavity samples from children with fever than in those without fever, and DNA of a broad range of viral species was detected [16▪]. Many studies have recently been performed on the function of the intestinal microbiome in Crohn's disease [17▪], ulcerative colitis [18▪], and esophageal cancer, function of the skin microbiome in psoriasis, atopic dermatitis, and immunodeficiency, function of the urogenital microbiome in pregnancy [19▪], sexual history, and surgery for phimosis, and function in many childhood diseases, such as pediatric abdominal pain and enteritis and the serious condition of premature babies with intestinal dysfunction [20▪]. There are still many unclear points with regard to the microbiome function in CRS, but allergic diseases and asthma are essential factors that need to be investigated regarding the development of CRS, and to understand the association between allergic diseases and asthma and the microbiome function, it is very important to elucidate the pathology of CRS.
The hygiene hypothesis is very important to consider the development of allergic diseases and asthma. In 1989, Strachan proposed that a lack of exposure to microbial environments in childhood because of the ‘clean environment’ accompanying the advancement in hygiene leads to the poor development of immunity and increases the risk of allergic diseases . This hygiene hypothesis is based on the observation that the risk of allergic diseases is low in children with many siblings and those who have grown up on farms with much contact with domestic animals. However, no direct relationship between infection and allergy was shown at that time, and this was a simplified observation. Later, a lower incidence of allergic and autoimmune diseases in individuals with numerous exposures to parasites was demonstrated by epidemiological and experimental studies . In 1998, it was clarified that changes in the intestinal bacterial composition in childhood caused by changes in lifestyle influence the tolerability of mucosal immunity and induce ‘distorted’ immune reactions, connecting the hygiene hypothesis with the relationship with microorganisms . Two cross-sectional surveys have recently been performed in a total of 16 000 children, and it was clarified that the development of asthma was inhibited in children with a more marked exposure to bacteria and fungi [24▪▪].
ALLERGIC DISEASES/ASTHMA AND INTESTINAL BACTERIA
Putting it simply, immunotolerance represents the biological capacity to identify antigens and exhibit defensive actions against these. Collapse of this tolerance is closely involved in the pathogenesis of various diseases. Acquired immunity plays an important role in the differentiation of self and nonself, but intestinal bacteria contain abundant nonself antigens including those derived from food regulating the body, and constantly present antigens to acquired immunity. Symbiotic microorganisms in the intestine develop a mechanism of inhibiting unnecessary inflammation by inducing immunotolerance through coordination with the natural immune system or regulation of the acquired immune system .
Allergic diseases and asthma are considered to be induced by excess reactions of Th2 cells. Th2 cells are characterized by IL-4, IL-5, IL-9, and IL-13 production, and these cytokines form and modulate the pathology of allergic inflammation. Not only Th2 cells but also Th1 cells are involved in the pathogenesis of asthma . Studies have shown the increasing role of Th17 and Th9 cells in asthma [27–29]. Tregs are important for the regulation of immunotolerance and play an important role in the modulation of inflammatory reactions [30–32].
Toll-like receptors and nucleotide binding oligomerization domain-like receptors are expressed in the intestinal epithelium, and dendritic cells are activated through these and regulate immunotolerance. It has been shown that intestinal bacteria, such as Lactobacillus and Bacteroides, promote Treg expression in the body and enhance the secretion of IL-10 and TGF-β [33,34]. Furthermore, it has been clarified that the oral ingestion of bacteria corrects the Th1/Th2 balance  and promotes differentiation into Th17 cells, suggesting that Th17 cells are involved in defense against pathogens reaching the intestine . The close involvement of these immunocytes in changes in intestinal bacteria has been shown, indicating that the intestinal bacterial balance is strongly associated with the development of allergic diseases and asthma.
EFFECTS OF MICROBIOME ON CHRONIC RHINOSINUSITIS
It has been clarified that various factors are involved in the pathogenesis of CRS [37▪]. For example, the relationship between CRS with nasal polyps (CRSwNP) and IgE for Staphylococcus aureus super antigen, as well as Th1/Th2 imbalance due to a decrease in Tregs is widely known. In addition, CRSwNP is closely associated with the development of asthma, based on which it is easy to imagine that the intestinal bacterial balance is involved in the development of CRS. However, there has been no report on the association between CRS and the intestinal bacterial microbiome. The association between CRS and bacteria in the nasal cavity and paranasal sinuses has been investigated in many studies, but gene analysis of these bacteria, that is, the microbiome, has only been performed in only a few studies [38–40,41▪▪]. The first reported study was performed in 2003, in which a bacteria-specific gene, 16s rDNA, was amplified from the mucosa and maxillary sinus lavage of 11 patients with maxillary sinusitis. Bacterial genes were amplified in four patients and identified as S. aureus, gram-positives, gram-negatives, and anaerobes. However, no fungus was detected. Later, two articles were published in 2010. In one article, bacterial gene analysis was performed using the mucosal samples. Bacterial genes were amplified in all 18 patients, and S. aureus and a lot of Coagulase-negative staphylococci were detected in many samples, but more anaerobes were detected. The involvement of anaerobes in the development of CRS has been suggested, and this study demonstrated it at the gene level. In contrast, maxillary sinus lavage was analyzed, and a total of 142 bacterial genes were amplified including many genes of indigenous bacteria in the oral cavity. In a recently reported study, cotton swabs of 15 CRS patients were analyzed, and more than 50 000 bacterial genes were detected in total. It was clarified that the incidence of asthma and the association with the diversity of the S. aureus gene increased as the diversity of bacterial genes in the samples increased. The relationship with S. aureus super antigen, which is considered to be the cause of CRS, and the development of asthma is also assumed on the basis of these viewpoints.
In the present review, we outlined existing information on CRS, allergic diseases, asthma, and the microbiome. There is still much to do in order to improve our understanding of the role this factor plays in disease development with regard to CRS. We need to understand the relationship of CRS not only with the intestinal bacterial microbiome but also with the microbiome in the nasal cavity and paranasal sinuses better, because of the difficulty in collecting samples from the paranasal sinuses without contamination, and inconsistency of the analytical methods due to the fact that no sampling methods have been established. As the recent progression in analytical methods has facilitated the investigation of microbiomes, further studies investigating the relationship between CRS and asthma in regards to the microbiome will be warranted.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 120–121).
1. Benninger MS, Ferguson BJ, Hadley JA, et al. Adult chronic rhinosinusitis: definitions, diagnosis, epidemiology, and pathophysiology. Otolaryngol Head Neck Surg 2003; 129:S1–S32.
2. Anand VK. Epidemiology and economic impact of rhinosinusitis. Ann Otol Rhinol Laryngol Suppl 2004; 193:3–5.
3. Senior BA, Glaze C, Benninger MS. Use of the Rhinosinusitis Disability Index (RSDI) in rhinologic disease. Am J Rhinol 2001; 15:15–20.
4. Lund VJ. Impact of chronic rhinosinusitis on quality of life and healthcare expenditure. Clin Allergy Immunol 2007; 20:15–24.
5. Kern RC, Conley DB, Walsh W, et al. Perspectives on the etiology of chronic rhinosinusitis: an immune barrier hypothesis. Am J Rhinol 2008; 22:549–559.
6. Van Cauwenberge P, Van Hoecke H, Bachert C. Pathogenesis of chronic rhinosinusitis. Curr Allergy Asthma Rep 2006; 6:487–494.
7. Van Bruaene N, Perez-Novo CA, Basinski TM, et al. T-cell regulation in chronic paranasal sinus disease. J Allergy Clin Immunol 2008; 121:1435–1441.
8. Lane AP, Truong-Tran QA, Schleimer RP. Altered expression of genes associated with innate immunity and inflammation in recalcitrant rhinosinusitis with polyps. Am J Rhinol 2006; 20:138–144.
9. Ramanathan M Jr, Lee WK, Spannhake EW, et al. Th2 cytokines associated with chronic rhinosinusitis with polyps down-regulate the antimicrobial immune function of human sinonasal epithelial cells. Am J Rhinol 2008; 22:115–121.
10. Ramanathan M Jr, Spannhake EW, Lane AP. Chronic rhinosinusitis with nasal polyps is associated with decreased expression of mucosal interleukin 22 receptor. Laryngoscope 2007; 117:1839–1843.
11. Schleimer RP, Kato A, Kern R, et al. Epithelium: at the interface of innate and adaptive immune responses. J Allergy Clin Immunol 2007; 120:1279–1284.
12. Schleimer RP, Lane AP, Kim J. Innate and acquired immunity and epithelial cell function in chronic rhinosinusitis. Clin Allergy Immunol 2007; 20:51–78.
13. Fokkens W, Lund V, Mullol J. EP3OS 2007: European position paper on rhinosinusitis and nasal polyps 2007: a summary for otorhinolaryngologists. Rhinology 2007; 45:97–101.
14. Eckburg PB, Bik EM, Bernstein CN, et al. Diversity of the human intestinal microbial flora. Science 2005; 308:1635–1638.
15▪▪. The Human Microbiome Project Consortium. A framework for human microbiome research. Nature 2012; 486:215–221
The newest findings of HMP project. From 242 adults, 5177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence have been generated.
16▪. Wylie KM, Mihindukulasuriya KA, Sodergren E, et al. Sequence analysis of the human virome in febrile and afebrile children. PLoS ONE 2012; 7:e27735.
On average nasopharynx and plasma samples from febrile children contained 1.5-fold to 5-fold more viral sequences, respectively, than samples from afebrile children.
17▪. Wu GD, Chen J, Hoffmann C, et al. Linking long-term dietary patterns with gut microbial enterotypes. Science 2011; 334:105–108.
Fecal communities clustered into enterotypes distinguished primarily by levels of bacteroides and prevotella. Enterotypes were strongly associated with long-term diets.
18▪. Zella GC, Hait EJ, Glavan T, et al. Distinct microbiome in pouchitis compared to healthy pouches in ulcerative colitis and familial adenomatous polyposis. Inflamm Bowel Dis 2011; 17:1092–1100.
The pouch microbial environment appears to be distinctly different in the settings of ulcerative colitis associated pouchitis, healthy ulcerative colitis, and familial adenomatous polyposis.
19▪. Ravel J, Gajer P, Abdo Z, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci USA 2011; 108 (Suppl 1):4680–4687.
The proportions of each community group varied among the four ethnic groups, Lactobacillus iners, L. crispatus, L. gasseri, or L. jensenii and these differences were statistically significant.
20▪. Mai V, Young CM, Ukhanova M, et al. Fecal microbiota in premature infants prior to necrotizing enterocolitis. PLoS One 2011; 6:e20647.
Abnormal patterns of microbiota and potentially a novel pathogen contribute to the cause of Necrotizing Entero Colitis.
21. Strachan DP. Hay fever, hygiene, and household size. BMJ 1989; 299:1259–1260.
22. Maizels RM. Exploring the immunology of parasitism – from surface antigens to the hygiene hypothesis. Parasitology 2009; 136:1549–1564.
23. Wold AE. The hygiene hypothesis revised: is the rising frequency of allergy due to changes in the intestinal flora? Allergy 1998; 53:20–25.
24▪▪. Ege MJ, Mayer M, Normand AC, et al. Exposure to environmental microorganisms and childhood asthma. N Engl J Med 2011; 364:701–709.
It was clarified that the development of asthma was inhibited in children with more marked exposure to bacteria and fungi.
25. Lee YK, Mazmanian SK. Has the microbiota played a critical role in the evolution of the adaptive immune system? Science 2010; 330:1768–1773.
26. Kero J, Gissler M, Hemminki E, et al. Could TH1 and TH2 diseases coexist? Evaluation of asthma incidence in children with coeliac disease, type 1 diabetes, or rheumatoid arthritis: a register study. J Allergy Clin Immunol 2001; 108:781–783.
27. Molet S, Hamid Q, Davoine F, et al. IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines. J Allergy Clin Immunol 2001; 108:430–438.
28. Cheng G, Arima M, Honda K, et al. Antiinterleukin-9 antibody treatment inhibits airway inflammation and hyperreactivity in mouse asthma model. Am J Respir Crit Care Med 2002; 166:409–416.
29. Wang YH, Voo KS, Liu B, et al. A novel subset of CD4(+) T(H)2 memory/effector cells that produce inflammatory IL-17 cytokine and promote the exacerbation of chronic allergic asthma. J Exp Med 2010; 207:2479–2491.
30. Karlsson MR, Rugtveit J, Brandtzaeg P. Allergen-responsive CD4+CD25+ regulatory T cells in children who have outgrown cow's milk allergy. J Exp Med 2004; 199:1679–1688.
31. Provoost S, Maes T, van Durme YM, et al. Decreased FOXP3 protein expression in patients with asthma. Allergy 2009; 64:1539–1546.
32. Wei W, Liu Y, Wang Y, et al. Induction of CD4+CD25+Foxp3+IL-10+ T cells in HDM-allergic asthmatic children with or without SIT. Int Arch Allergy Immunol 2010; 153:19–26.
33. Round JL, Mazmanian SK. Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proc Natl Acad Sci USA 2010; 107:12204–12209.
34. Ly NP, Ruiz-Perez B, Onderdonk AB, et al. Mode of delivery and cord blood cytokines: a birth cohort study. Clin Mol Allergy 2006; 4:13.
35. Ghadimi D, Folster-Holst R, de Vrese M, et al. Effects of probiotic bacteria and their genomic DNA on TH1/TH2-cytokine production by peripheral blood mononuclear cells (PBMCs) of healthy and allergic subjects. Immunobiology 2008; 213:677–692.
36. Ivanov II, Atarashi K, Manel N, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 2009; 139:485–498.
37▪. Van Crombruggen K, Zhang N, Gevaert P, et al. Pathogenesis of chronic rhinosinusitis: inflammation. J Allergy Clin Immunol 2011; 128:728–732.
This review focuses on recent evidence that sheds new light on our current knowledge regarding the inflammatory process of CRS to further unravel its pathogenesis.
38. Paju S, Bernstein JM, Haase EM, et al. Molecular analysis of bacterial flora associated with chronically inflamed maxillary sinuses. J Med Microbiol 2003; 52:591–597.
39. Stephenson MF, Mfuna L, Dowd SE, et al. Molecular characterization of the polymicrobial flora in chronic rhinosinusitis. J Otolaryngol Head Neck Surg 2010; 39:182–187.
40. Lewenza S, Charron-Mazenod L, Cho JJ, et al. Identification of bacterial contaminants in sinus irrigation bottles from chronic rhinosinusitis patients. J Otolaryngol Head Neck Surg 2010; 39:458–463.
41▪▪. Feazel LM, Robertson CE, Ramakrishnan VR, et al. Microbiome complexity and Staphylococcus aureus in chronic rhinosinusitis. Laryngoscope 2012; 122:467–472.
This is the first report in which bacterial DNA in the nose was analyzed by pyrosequence. Greater abundance of S. aureus may characterize the disease state of CRS.
© 2013 Lippincott Williams & Wilkins, Inc.