Utilization of Transcriptomic Profiling to Identify Molecular Markers Predicting Successful Recovery Following Endoscopic Sinus Surgery for Chronic Rhinosinusitis.

OBJECTIVES: Successful recovery from chronic rhinosinusitis (CRS) following endoscopic sinus surgery (ESS) can be characterized by minimal presence of symptoms and absence of disease on endoscopy. However, molecular markers of surgical success remain to be characterized. These could allow for better tailoring of perioperative therapy. This study aims to identify novel molecular markers associated with surgery responsive patient. STUDY DESIGN: Prospective cohort study. SETTING: Single academic hospital center. METHOD: One hundred eighteen consecutive patients with CRS at high risk of recurrence after surgery were followed prospectively following ESS in an academic medical center. Symptomatic and endoscopic outcomes were assessed at 4 months, with success rigorously defined subjectively as minimal or no symptoms (no symptom greater than 1 on an ordinal scale of 0-3) and objectively by the absence of nasal polyposis on sinus cavity endoscopy and Lund-Kennedy endoscopic edema score no greater than 1. Samples were obtained at the time of surgery and at 4-month postoperatively. Changes associated with surgery were determined by gene expression profiling using Affymetrix's Clariom S Human HT arrays. RESULTS: Successful ESS was characterized by a mild upregulation in Type 1 inflammation, upregulation of cell cycle progression, and epithelial barrier and proliferation-associated genes and pathways. ESS failure was associated to very high levels of Type 1 inflammation along with downregulation of epithelial barrier function and regeneration genes and pathways. CONCLUSION: Successful recovery from ESS involves restoration of epithelial function and regulated activation of Type 1 inflammation. Excessively elevated Type 1 inflammation is associated with epithelial barrier dysfunction.

Azithromycin Mechanisms of Action in CRS Include Epithelial Barrier Restoration and Type 1 Inflammation Reduction.

OBJECTIVE: Previous in vitro transcriptomic profiling suggests azithromycin exerts its effects in patients with chronic rhinosinusitis (CRS) via modulation of type 1 inflammation and restoration of epithelial barrier function. We wished to verify these postulated effects using in vitro models of epithelial repair and in vivo transcriptional profiling. STUDY DESIGN: Functional effects of azithromycin in CRS were verified using in vitro models of wounding. The mechanism of the effect of azithromycin was assessed in vivo using transcriptomic profiling. SETTING: Academic medical center. METHODS: Effects of azithromycin on the speed of epithelial repair were verified in a wounding model using primary nasal epithelial cells (pNEC) from CRS patients. Nasal brushings collected pre-and posttreatment during a placebo-controlled trial of azithromycin for CRS patients unresponsive to surgery underwent transcriptomic profiling to identify implicated pathways. RESULTS: Administration of azithromycin improved the wound healing rates in CRS pNECs and prevented the negative effect of Staphylococcus aureus on epithelial repair. In vivo, response to azithromycin was associated with downregulation in pathways of type 1 inflammation, and upregulation of pathways implicated in the restoration of the cell cycle. CONCLUSION: Restoration of healthy epithelial function may represent a major mode of action of azithromycin in CRS. In vitro models show enhanced epithelial repair, while in vivo transcriptomics shows downregulation of pathways type 1 inflammation accompanied by upregulation of DNA repair and cell-cycle pathways. The maximal effect in patients with high levels of type 1-enhanced inflammation suggests that azithromycin may represent a novel therapeutic option for surgery-unresponsive CRS patients.

Response to intranasal Lactococcus lactis W136 probiotic supplementation in refractory CRS is associated with modulation of non-type 2 inflammation and epithelial regeneration.

Justification: We have previously documented that in individuals with chronic rhinosinusitis (CRS) refractory to surgery, intranasal application of live Lactococcus lactis W136, a probiotic bacterium, improves sinus-specific symptoms, SNOT-22, and mucosal aspect on endoscopy, accompanied by a reduction in sinus pathogens and an increase in protective bacteria. The present work explores the molecular mechanisms underpinning these observations using transcriptomics of the sinus mucosa. Method: Epithelial brushings collected prospectively as a sub-study of the L. lactis W136 clinical trial were used to probe epithelial responses to microbiome supplementation using a hypothesis-free bioinformatic analysis of gene expression analysis. Samples from twenty-four patients with CRS refractory to medical and surgical management were prospectively collected during a clinical trial assessing the effect of 14 days of BID nasal irrigation with 1.2 billion CFU of live L. lactis W136 probiotic bacteria (CRSwNP = 17, CRSsNP = 7). Endoscopically guided sinus brushings were collected as part of the initial study, with brushings performed immediately before and after treatment. Following RNA extraction, samples were assessed using the Illumina HumanHT-12 V4 BeadChip. Differential gene expression was calculated, and pathway enrichment analysis was performed to identify potentially implicated processes. Results: Differentially identified transcripts and pathways were assessed for the overall population and the clinical phenotypes of CRSwNP and CRSsNP. Patterns of response to treatment were similar across all groups, implicating pathways for the regulation of immunity and epithelial cell regulation. These resemble the patterns of improvement observed following successful treatment with endoscopic sinus surgery or azithromycin. Conclusion: Gene expression profiling following the application of live bacteria to the diseased sinus epithelium highlights the implication of multiple components of the inflammation-microbiome-epithelial barrier axis implicated in CRS. These effects appear to involve both epithelial restoration and modulation of innate and adaptive immunity, supporting the potential interest of targeting the sinus epithelium and the microbiome as potential CRS therapies.

Low-dose and long-term azithromycin significantly decreases Staphylococcus aureus in the microbiome of refractory CRS patients.

BACKGROUND: The sinonasal microbiome is believed to play an important role in the pathophysiology of refractory chronic rhinosinusitis (CRS). We evaluated changes in the microbiome following a 4-month course of low-dose azithromycin. Assessing microbiome alterations following such a treatment may help identify underlying mechanisms of this drug. METHODS: A total of 48 adults with refractory CRS were enrolled in a double-blind, randomized, placebo-controlled trial. Patients were randomized to 250 mg of azithromycin or placebo 3 times weekly for 4 months. During this time, daily budesonide saline irrigations were continued. Sinonasal swabs were collected by endoscopically-assisted method prior to treatment initiation and at the end of it, and sent for 16S ribosomal RNA gene sequencing. High-resolution ANCHOR pipeline was used to infer and annotate putative species. The 2 patient groups were compared using DESeq2 differential abundance analysis. RESULTS: From initiation to the end of azithromycin treatment, patients showed a significant difference in beta diversity analysis (p = 0.0004) along with a significant decrease in 71 different operational taxonomic units (OTUs) of Staphylococcus aureus (false discovery rate [FDR] < 0.05) obtained from the differential abundance analysis. This was not observed in placebo-treated patients. By the end of treatments, azithromycin-treated patients had a significant decrease in 29 different OTUs of S. aureus (FDR < 0.05) when compared to placebo. CONCLUSION: A 4-month course of 250 mg of azithromycin 3 times weekly in patients with refractory CRS significantly decreases S. aureus abundance in the sinonasal microbiome. Considering the pathogenic role of S. aureus in the refractory CRS population, azithromycin may constitute an additional therapeutic option to help control this disease.

Molecular characterization of the polymicrobial flora in chronic rhinosinusitis.

INTRODUCTION: Conventional cultures have implicated Staphylococcus aureus (SA) and coagulase-negative Staphylococcus (CNS) as principal pathogens in chronic rhinosinusitis (CRS). These results are questioned by recent studies in which molecular probes implicate Haemophilus influenzae instead. OBJECTIVES: To identify all bacterial species present on sinonasal mucosa using molecular culture (bacterial tag-encoded FLX amplicon pyrosequencing [bTEFAP]) and to compare them with those identified with conventional methods. METHODS: A prospective study of 18 patients undergoing endoscopic sinus surgery for CRS and 9 control patients with pituitary adenomas was conducted. Per-operative mucosal biopsies were assessed with bTEFAP by sequencing the species-specific 16S ribosomal deoxyribonucleic acid (DNA) fragment for genetic identification of bacteria and then compared with simultaneous swab culture. RESULTS: Standard cultures showed mainly SA and CNS. Molecular cultures identified up to 20 organisms per sample. Surprisingly, anaerobic species predominated (Diaphorobacter and Peptoniphilus). SA was nevertheless detected in 50%. CONCLUSION: Molecular cultures such as bTEFAP are sensitive tools for bacterial identification in CRS and suggest that anaerobe involvement may be more frequent than presumed.

A pooling-based genome-wide association study implicates the p73 gene in chronic rhinosinusitis.

BACKGROUND: p73 is a gene that may confer a genetic susceptibility to the development of chronic rhinosinusitis. OBJECTIVE: To verify an association between p73 and chronic rhinosinusitis, identified in a pooling-based genome-wide association study in a human population. METHODS: Prospective recruitment of 206 patients and 196 postal code-matched controls. DNA extraction, followed by pooling, high-density single nucleotide polymorphism (SNP) genotyping, and individual genotyping, was carried out. Gene sequencing was carried out in 10 patients and 1 control. RESULTS: Statistical analysis of SNP rs3765731 revealed a significant difference in minor allele frequency between the case and control groups. Minor allele A was more frequent in the healthy group, with an odds ratio of 0.6533, whereas G had a higher association with the disease state. Subgroup analysis of allele frequencies showed A to be associated with a lesser likelihood of developing chronic rhinosinusitis. Homozygous AA patients with severe disease had a sevenfold lesser risk of chronic rhinosinusitis compared with GG homozygotes (odds ratio 0.14). Sequencing did not reveal any amino acid changes conferring a change of function in secreted proteins. CONCLUSION: We have demonstrated an association between rs3765731 and chronic rhinosinusitis. Minor allele A has a protective effect compared with major allele G. Interaction with other signaling proteins and induction of other genes will better explain this mechanism.

Polymorphisms in the tumour necrosis factor alpha-induced protein 3 (TNFAIP3) gene are associated with chronic rhinosinusitis.

BACKGROUND: Factors conferring susceptibility to chronic rhinosinusitis remain unknown. However, advances in genomics offer powerful tools to explore this disorder. Tumour necrosis factor (TNF) is a crucial proinflammatory cytokine that exerts inflammatory and immunomodulatory activities important in host defense. Our objective was to determine whether polymorphisms in genes in the TNF superfamily (TNF, TNF-alpha-induced protein 3, TNF-alpha-induced protein 6) were associated with chronic rhinosinusitis. METHODS: Deoxyribonucleic acid (DNA) extracted from a population of 206 patients with severe chronic rhinosinusitis and 196 postal code-matched controls was used. Three candidate genes related to the TNF inflammatory pathway were assessed. For each gene, an informative set of single nucleotide polymorphisms was genotyped. RESULTS: Thirty-five single nucleotide polymorphisms were genotyped. Two polymorphisms located within the TNF-alpha-induced protein 3 gene (TNFAIP3) reached the nominal p value threshold (p < .05) for association with chronic rhinosinusitis. However, none of these polymorphisms resist multiple testing adjustments. CONCLUSIONS: Our data suggest that two polymorphisms in TNFAIP3 are weakly associated with severe chronic rhinosinusitis but do not support an association with genetic variants in TNF or TNF-alpha-induced protein 6. Although these results do not support correction for multiple testing and have to be validated in a second population, they nevertheless suggest that further studies of the role of TNFAIP3 in the pathogenesis of disease are warranted.