THE EFFECT OF MEDICAL TREATMENTS ON THE BACTERIAL MICROBIOME IN PATIENTS WITH CHRONIC RHINOSINUSITIS

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CHAPTER 2. DIFFERENCES IN THE PARANASAL SINUSES BETWEEN GERM–FREE AND PATHOGEN–FREE MICE.

Abstract

Background

The role of bacteria in the aetiology of chronic rhinosinusitis (CRS) is not fully understood. Commensal bacteria may have a significant impact on the development of normal paranasal sinus anatomy and mucosal immunity, as they do in the gut. Studying the paranasal sinuses of germ-free mice (GFM) may provide some insight into the effect of commensal bacteria on sinus structure and mucosal function.

Methods

The paranasal sinuses of five GFM were compared to five pathogen-free normal mice. Mice heads underwent computed tomography and images compared for pneumatisation and geometry of the sinuses. Histologically, slides were examined by light microscopy and compared for mucosal thickness, epithelial thickness, cilia, collagen, goblet cells and nasal-associated lymphatic tissue (NALT).

Results

No radiological differences were seen between groups. Overall, GFM were found to have thinner mucosa (D 15.2 ± 5.2 µm, p = 0.004), thinner epithelium (D 5.5 ± 2.6 µm, p = 0.037), more collagen (D 5.8 ± 1.6 %, p < 0.001), fewer goblet cells (D 29.3 ± 5.4, p < 0.001) and less NALT (D 14900 ± 6700 µm², p = 0.04). Sub-analysis by region revealed significant differences for GFM in the middle (thinner mucosa, thinner epithelium, fewer cilia and more collagen) and posterior (fewer goblet cells) sinus sections.

Conclusions

The results of this study demonstrate that commensal microbiota significantly contribute to the structure and function of murine paranasal sinuses. Therefore, changes in commensal microbiota associated with CRS may alter the normal microbe host dialogue in humans and be implicated in the pathogenesis of CRS

Introduction

Complex microbial communities have been observed in the paranasal sinuses of patients with chronic rhinosinusitis (CRS). Bacteria exist in planktonic states, structured biofilms and within the mucosa in small intracellular and extracellular colonies. However, the influence of these communities on the precipitation and perpetuation of CRS remains unknown.
The interactions between the microbiota, the innate immune system and mucosal tissue are not well understood. Elucidation of these interactions may help guide the development of new clinical approaches to CRS.
The relationship between bacteria and gastrointestinal mucosa has been described in germ free animal models.^65,66 These studies of the intestines of germ free mice have found reduced intestinal hydration, less lamina propria, a reduced total mucosal surface area and down regulation of the mononuclear phagocyte system. Further studies have credited commensal bacteria in the intestine with the control of inflammation that normally exists in health by affecting expression of host defence components, which promote development of intestinal innate immunity and local immunity.^67-69 Inflammatory bowel disease has been suggested to occur due to an altered abnormal response to commensal antigens rather than a normal immunological response to specific pathogens. Accordingly, it is believed that indigenous gut bacteria are centrally involved in the development of inflammatory bowel disease.⁷⁰ Recently, complex microbial communities have been described in the sinuses of patients with and without CRS. These studies have suggested a shift in disease towards a reduction of species diversity and richness.⁷¹ To date, it remains unclear how this dysbiosis is acquired and what the implications are for improving clinical outcomes, including post-operative mucosal healing.
The aim of this study was to describe the relationship between host microbiota and the functional components of mucosal tissue by comparing histological differences in the paranasal sinuses between germ free (animals free of microbial colonisation) and specific pathogen-free mice (animals with normal commensal microbiota but free of a wide range of pathogens)

Subjects and methods

In this study five germ-free (GF) and five conventional, specific pathogen-free (SPF) mice were compared. Germ-free and SPF mice (C3H/Orl, Charles River Laboratories International, Saint-Germain-Nuelles, France) were maintained in isolators at the Royal Veterinary College, University of London, or in individually ventilated cages at the animal care facilities of Queen 9 Mary University of London respectively. Animal care was approved by the institutional animal ethics committee.
The sterility of GF animals was examined by aerobic and anaerobic culture of oral swabs and faecal pellets on non-selective media and by PCR using universal 16S primers. Studies further describing the sterility of this GF colony and which bacteria have been isolated from the SPF mice have been previously published.^72-75
Mice were decapitated and fixed in fresh Carnoy’s solution at University of London before being sent to The University of Auckland for radiological and histological analysis. All animals were 10-12 week old males and euthanised following five minutes of carbon dioxide asphyxiation

Radiological analysis

All mice heads underwent computed tomography (Skyscan-1172, Bruker microCT, Kontich, Belgium). Images were acquired in 35 µm slices in coronal and sagittal planes. Images were compared at the levels of the superior tooth, the anterior aspect of the secondary maxillary sinus, the anterior aspect of the true maxillary sinus, the ethmoid sinuses at the level of the orbits and the sphenoid sinuses at the most anterior aspect of the brain. (Figure 2.1) Measurement of the maximal sinus width was taken at each level for each animal using ImageJ (National Institute of Health, Bethesda, MD; http://imagej.nih.gov/ij) and compared between the mice groups by level and overall average

Tissue Preparation

The mice heads were decalcified in 10% formic acid for one week before being transferred to 70% ethanol. The mice snouts were divided into three (approx. 3 mm) sections through the paranasal sinuses – representing an anterior segment, middle segment and posterior segment. These segments were processed and embedded in paraffin. Sections were cut on a microtome at 5 µm. Multiple slides were prepared and stained with Sirius red-light green, periodic acid Schiff-Alcian blue, immunohistochemical and hematoxylin and eosin stains. (Figure 2.1)

Histological analysis

Slides were prepared for each of the three regions of each mouse. Sections were analysed independently by two authors (RJ and SWT), who were blinded to mouse group. Slides from each region stained with Sirius red-light green were digitally slide scanned (MetaSystems GmbH, Altlussheim, Germany) and analysed for cilia, collagen content, mucosal thickness and epithelial thickness in ImageJ (National Institute of Health, Bethesda, MD; http://imagej.nih.gov/ij) A total of eight regions were measured for each slide (four from the left side and four from the right). Epithelial and mucosal thicknesses were measured using calipers following scale calibration in ImageJ. Collagen was calculated as a percentage of the full-thickness mucosal tissue following colour deconvolution and thresholding for collagen in ImageJ. The presence of cilia was graded on a subjective scale from 0 (absent cilia) to 3 (full brush border). The total numbers of goblet cells per slide were counted on sections stained with periodic acid Schiff–Alcian blue.
Lymphocytes were analysed in the regions of nasal-associated lymphatic tissue (NALT), which were seen in the posterior sections of each mouse using immunohistochemistry. Five micrometer sections from each mouse were stained with CD3 1:300 (Leica Biosystems, Nussloch, Germany) and CD45R/B220 Biotin 1:20 (BD Biosciences, Franklin Lakes, NJ) for 4 degrees overnight. This was followed by either the Novalink Polymer Detection System (Leica Biosystems, Nussloch, Germany) or Streptavadin HRP (NEN, Boston, MA) respectively and diaminobenzidene (DAB).
ImageJ was used to perform colour deconvolution on NALT regions to isolate the brown channel for DAB positivity and then calculate areas of positivity as a percentage of total NALT area

 Statistical analysis

Statistical analyses were performed using SPSS (Version 22; IBM, New York). The difference between pooled mean scores of the GF and SPF mice were compared using a 2-tailed Student’s t-test. Statistical significance for all tests was accepted when p < 0.05. Results are presented as mean ± standard error.

Results

Radiology

No significant differences were seen between GF mice and SPF mice when CT images were compared by overall group average (D 0.008 ± 0.09 µm, p = 0.9). Further analysis by sublevels showed no significant differences at all levels – superior tooth (D 0.03 ± 0.05 µm, p = 0.5), the secondary maxillary sinus (D 0.006 ± 0.06 µm, p = 0.9), the true maxillary sinus (D 0.08 ± 0.08 µm, p = 0.4), the ethmoid sinuses (D 0.05 ± 0.06 µm, p = 0.5) and the sphenoid sinuses (D 0.03 ± 0.08 µm, p = 0.7).

Histology

Initially the scores from all sites and regions were pooled and results between GF mice and SPF mice compared. (Table 2.1) Respiratory epithelium was found in the anterior and middle sections, while the posterior section was exclusively olfactory epithelium in both groups of mice.
Overall differences were seen for mucosal thickness (D 15.2 ± 5.2 µm, p = 0.004), epithelial thickness (D 5.5 ± 2.6 µm, p = 0.04), tissue collagen (D 5.8 ± 1.6 %, p < 0.001) and total number of goblet cells (D 29.3 ± 5.4, p < 0.001). No significant difference in cilial score was seen between the two groups (D 0.1 ± 0.06). (Table 2.1, Figures 2.2 and 2.3)
Sub-analysis by region (Table 2.2) demonstrated differences for mucosal thickness in the middle segment (D 33 ± 11 µm, p = 0.004), epithelial thickness in the middle segment (D 10 ± 1.9 µm, p 001), cilial scores in the middle segment (D 0.3 ± 0.1, p = 0.003), tissue collagen in the anterior and middle segments (D 12.0 ± 3.2 %, p < 0.001 and 5.6 ± 1.5 %, p = 0.001) and goblet cells in the anterior and posterior segments (D 68 ± 13, p < 0.001 and 16 ± 2.6, p < 0.001). (Table 2.2)
The overall area of NALT tissue was significantly less in GF animals (D 14900 ± 6700 µm², p = 0.04). (Figure 2.4) On average GF mice had slightly fewer CD3 (D 5.7 ± 6.1 %, p = 0.4) and fewer CD45R/B220 (D 2.2 ± 7.9 %, p = 0.8) than SPF animals, although this was not significant for either group. Macrophages were not present in sufficient numbers in any tissue sections for analysis

CHAPTER 1. INTRODUCTION.
1.1 Disease burden
1.2 Pathophysiology
1.3 Medical therapy
1.4 Endoscopic sinus surgery
1.5 Hypotheses
CHAPTER 2. DIFFERENCES IN THE PARANASAL SINUSES BETWEEN GERM-FREE AND PATHOGEN-FREE MICE. 
2.1 Abstract
2.2 Introduction
2.3 Subjects and methods
2.4 Results
2.5 Discussion
2.6 Conclusion
CHAPTER 3. THE EFFECT OF MEDICAL TREATMENTS ON THE BACTERIAL MICROBIOME IN PATIENTS WITH CHRONIC RHINOSINUSITIS: A PILOT STUDY. 
3.1 Abstract
3.2 Introduction
3.3 Methods
3.4 Results
3.5 Discussion
3.6 Conclusion
CHAPTER 4. THE IN VITRO EFFECT OF XYLITOL ON CHRONIC RHINOSINUSITIS BIOFILMS.
4.1 Abstract
4.2 Introduction
4.3 Methods
4.4 Results
4.5 Discussion
4.6 Conclusion
CHAPTER 5. THE IMPACT OF ENDOSCOPIC SINUS SURGERY ON PARANASAL PHYSIOLOGY IN SIMULATED SINUS CAVITIES. 
5.1 Abstract
5.2 Introduction
5.3 Methods
5.4 Results
5.5 Discussion
5.6 Conclusion
CHAPTER 6. CHANGES IN THE BACTERIAL MICROBIOME OF PATIENTS WITH CHRONIC RHINOSINUSITIS AFTER ENDOSCOPIC SINUS SURGERY. 
6.1 Abstract
6.2 Introduction
6.3 Methods
6.4 Results
6.5 Discussion
CHAPTER 7. THE EFFECTS OF TOPICAL AGENTS ON PARANASAL SINUS MUCOSA HEALING: A RABBIT STUDY. 
7.1 Abstract
7.2 Introduction
7.3 Subjects and methods
7.4 Results
7.5 Discussion
7.6 Conclusion
CHAPTER 8. GENERAL DISCUSSION AND CONCLUSIONS
8.1 Major findings
8.2 Discussion
8.3 Future directions
8.4 Conclusion
CHAPTER 9. BIBLIOGRAPHY.
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