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Original Investigation |

Contrasting the Microbiomes From Healthy Volunteers and Patients With Chronic Rhinosinusitis

Rajeev Aurora, PhD1; Dhrubamitra Chatterjee, BSc1; Joshua Hentzleman, MD2; Gaurav Prasad, MD2; Raj Sindwani, MD3; Thomas Sanford, MD2
[+] Author Affiliations
1Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St Louis, Missouri
2Department of Otolaryngology, Saint Louis University School of Medicine, St Louis, Missouri
3Head and Neck Institute, Cleveland Clinic Foundation, Cleveland, Ohio
JAMA Otolaryngol Head Neck Surg. 2013;139(12):1328-1338. doi:10.1001/jamaoto.2013.5465.
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Published online

Importance  Chronic rhinosinusitis (CRS) is the persistent inflammation of the sinus and nasal passages lasting over 3 months. The etiology of CRS is not well understood.

Objective  To obtain insights into the disease process, we contrasted the microbiome and immune response from patients with CRS and healthy controls.

Design, Setting, and Participants  A case vs control design was used. Samples were collected in the operating room in an institutional hospital or clinic. Thirty patients with CRS and 12 healthy controls undergoing surgery were recruited.

Main Outcomes and Measures  The microbiome was analyzed by deep sequencing of the bacterial 16S and fungal 18S ribosomal RNA genes. Immune response was measured by quantification of 30 different cytokines by multiplexed enzyme-linked immunosorbent assay, and immune cells in the lavage were identified by flow cytometry. The immune response of peripheral blood leukocytes to the lavage microbiota was assessed by interleukin (IL)-5 enzyme-linked immunospot assay.

Results  While quantitative increase in most bacterial and fungal species was observed in patients with CRS relative to controls, the microbiomes of patients with CRS were qualitatively similar to the controls. Because these results indicated that bacteria and fungi are not triggering CRS, we undertook a more detailed characterization of the immune response. Patients with CRS had increased levels of the following cytokines: IL-4, IL-5, IL-8, and IL-13, along with increased levels of eosinophils and basophils in the lavage. Importantly, peripheral blood leukocytes isolated from patients with CRS responded to control lavage samples (ie, to commensals) to produce IL-5. In contrast, the same lavage sample evoked no IL-5 production in leukocytes from healthy controls.

Conclusions and Relevance  These findings support the theory that in some cases CRS results from an immune hyperresponsiveness to commensal organisms.

Figures in this Article

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Figures

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Figure 1.
The Bacterial Microbiome in Chronic Rhinosinusitis (CRS) and Control Lavage Samples

A, The overlap of all species identified by Roche 454 sequencing of the 16S ribosomal RNA (rRNA) amplicons is shown (values represent numbers of species). The patients with CRS had a greater diversity of organisms. Although 37% of the species overlap, the CRS- and control-specific sets are species present at very low abundance (<0.1%). Furthermore, these species were not present in all 30 CRS and 12 control lavage samples. B, The distribution of species (rank order vs number of raw reads for each of the species) is shown. The plot shows that 1 species was dominant and most species were present at less than 0.1% (<800 reads for a species/800 000 total reads). C, The number of species within each phylum and corresponding fraction of reads are shown for CRS and control samples. All species identified, regardless of distribution across patient samples, are given in this table. D, The species with the highest abundance in patients with CRS is shown, along with their fractional abundance in the control samples. Only those species that were found in all 29 patients with CRS are listed. P values were calculated using a 2-tailed Mann-Whitney test. NS indicates nonsignificance (P > .05). E, A pairwise comparison of the overlap of the (healthy) control sinus microbiome to the 20 topological skin areas sampled by Grice et al35 using the Yue-Clayton θ dissimilarity measure. The external auditory (Ext aud) canal had the highest similarity (lowest θ value) due in part to the abundance of Cyanobacteria and number of Actinobacteria species. Axill vault indicates axillary vault; RA crease, retroauricular crease.aIncreased abundance in patients with CRS relative to controls (panel D)bIncreased abundance in controls relative to patients with CRS (panel D).

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Figure 2.
The Fungal Microbiome in Chronic Rhinosinusitis (CRS) and Control Lavage Samples

A, The overlap of all species identified by deep sequencing of the 18S rRNA amplicons is shown (values represent numbers of species). The fungal diversity was increased in lavage samples from patients with CRS. As in the case of the bacterial microbiome, the abundance of the species unique to each set is very low (<0.1%), and the set-unique species were found in 1 or 2 patient or control samples. B, The distribution of species (ordered by rank) is shown. In both CRS and control samples, a single fungal species was dominant. C, The number of species in each phylum and the percentage of reads for the phylum are given. D, The most abundant species found in CRS and control lavage samples are shown. Cryptococcus neoformans variety neoformans (strain D) was the dominant species in both CRS and control sampling. P values were calculated using the 2-tailed Mann-Whitney test. NS indicates nonsignificance (P > .05).aIncreased abundance in patients with CRS relative to controls (panel D)bIncreased abundance in controls relative to patients with CRS (panel D).

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Figure 3.
Assessment of Cytokines From the Lavage of Patients With Chronic Rhinosinusitis (CRS) and Healthy Controls

Multiplexed enzyme-linked immunosorbent assay was used to measure 32 different cytokines in the lavage samples. Four cytokines (interleukin [IL]-4, IL-5, IL-8, and IL-13) showed statistically significant differences between CRS and control samples.

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Figure 4.
Characterization of the Immune Response Using Fluorescent-Activated Cell Sorter

A, The gating scheme for identification of the immune cells. B, The quantification of the immune cells. The numbers given in the “Controls” and “Patients With CRS” columns are the mean (SD) percentage of the parent cell population. For instance, 1.6% (0.8%) of CD3+ (T cells) were CD4+ T cells. P values in bold indicate statistical significance. CRS indicates, chronic rhinosinusitis; ND, not detected; and NK, natural killer.aIncreased abundance in controls relative to patients with CRS (panel B).bIncreased abundance in patients with CRS relative to controls (panel B).

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Figure 5.
Patients With Chronic Rhinosinusitis (CRS) are Hyperresponsive to Normal Sinus Microbiome

Ten thousand or 50 thousand peripheral blood leukocytes isolated from either 5 patients with CRS or 5 healthy control volunteers (NOR) were cocultured with lavage from the same control (nondiseased) individuals. The immune response was measured using an interleukin (IL)-5 enzyme-linked immunospot (ELISpot). Each data point is an average of 6 to 8 wells. Leukocytes from 1 patient and 1 control were cocultured with 2 different lavage pellets: 1 from self and 1 from a nonself lavage. The leukocytes from the patients with CRS produced 278.3 [50.3] (mean [SD]) IL-5 positive spots compared with leukocytes from healthy volunteers that produced 12.2 [5.8] spots (P = .005). Coculturing leukocytes from patient with CRS with lavage from patient with patients produced 350 [47.3]–positive ELISpots, compared with leukocytes from healthy volunteers cocultured with the same CRS lavage that produced 75.0  [34.8] IL-5–positive ELISpots. To test for IL-5 carryover from the CRS lavage samples, we also performed ELISpot assay from lavage alone, and we observed 3.3 (1.3) spots. P values were calculated using the 2-tailed Mann-Whitney test in Prism 5.0d statistical package (GraphPad). The dashes in the y-axis labels indicate that the component (leukocytes or microbiota) was omitted in those set of wells.

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Figure 6.
Microbiome, Host Response, and Disease

The microbiome is a spectrum of symbionts (mutually beneficial to microbe and host), commensals (neutral for microbe and host), and pathogens (detrimental to host) that has been described as mutualism.54 In addition to the mutualism axis shown on the x-axis, the immune response (y-axis) and disease severity (z-axis) are also drawn. The illustration is intended to show that the immune response keeps the levels of commensals in check as an appropriate immune response. In contrast, too strong an immune response to commensals can lead to inflammation and damage to host.

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