Download - Understanding the Role of Fungi in the Pathophysiology And

According to the Centers for Disease Control and Prevention (CDC), the

current prevalence of sinusitis in the US is up to 13.4% of the population

(29.5 million adults).1 A clear understanding of the underlying

pathophysiology and effective medical treatment options is still lacking for

chronic rhinosinusitis (CRS). Patients with CRS suffer from various

symptoms, including nasal congestion, copious mucus production, cough,

and acute exacerbations with facial pain, the latter presumably secondary to

bacterial infections.2 Furthermore, the socioeconomic burden of CRS is

profound considering that the current estimated number of office-based

visits is 12.5 million a year.1 Given the great impact of CRS, it is imperative

to better understand the mechanisms underlying the disease process so that

appropriate treatment can be employed.

Bacterial Infections in Chronic Rhinosinusitis

The fact that patients with CRS experience recurrent bacterial exacerbations

fueled the notion that the disease process is a bacterial infection resistant to

antibiotic intervention and surgery. However, bacterial infections always

produce a neutrophilic inflammation and, in contrast, the chronic

inflammation in CRS is predominantly mediated by eosinophils.3–6 Thus,

bacterial infections likely play a role in the acute exacerbations—when

neutrophils can also be identified—but it is unlikely that they induce the

underlying inflammatory process.

It Is All About Eosinophils

Eosinophils are typically associated with immunoglobulin E (IgE)-mediated

allergies. They are recruited into affected tissue by cytokines and

chemokines during the late phase of the allergic reaction. However, in CRS

only 40–60% of patients have a positive allergy test, and the eosinophilic

inflammation is present with the same intensity in those patients who have

detectable IgE-mediated allergies as in those that do not.7 Also patients with

IgE-mediated allergies develop a different symptom complex from those

with CRS, including sneezing and itching eyes. In other words, patients with

CRS who have elevated IgE levels may have allergic rhinitis as a comorbid

disease, but the mechanism behind the eosinophilic inflammation appears

to be independent of the IgE-mediated allergic response.

However, previous studies found that eosinophils were not present in all CRS

cases. This could have been due to the use of systemic corticosteroids prior

to surgery when tissue samples are collected. Steroids are potent inhibitors

of eosinophilic inflammation and can lead to the lack of eosinophils found in

these samples. In addition, the eosinophilic inflammation is extremely

heterogenous. It is absent in large areas of harvested specimen, only to be

present in abundance in other areas only millimeters away.8 Thus, by avoiding

pre-operative steroids and by sampling larger or multiple sites one can

appreciate better the eosinophilic nature of the inflammation in CRS.

Cytokines and Immunoglobulins

As explained above, CRS cannot be adequately explained by theories of IgE-

mediated hypersensitivity, nor is there sufficient evidence to show that CRS

is due to bacterial inflammation. However, a prominent IgE independent

and cytokine-driven eosinophilic inflammation has been found in these

patients. Interleukin (IL)-5, IL-13 (both TH-2), and interferon (IFN)-gamma

(TH-1) activate and prolong the lifespan of eosinophils, induce their

migration from the vasculature to sinus tissue, and enhance their destructive

ability, respectively.9–11 All of these cytokines are present in CRS tissue and

absent in healthy controls.12 In order to determine the causative agent

responsible for inciting the eosinophilic inflammation, one has to find the

trigger for the cytokine production.

An Immune Response to Fungi

To study whether certain fungi could incite the production of cytokines,

peripheral blood mononuclear cells (PBMCs) from CRS patients—which

a report by

Niv Mor , BA ,1 David A Sherr is , MD ,1 Hirohito Kita , MD 2 and Jens U Ponikau , MD 1

1. Department of Otorhinolaryngology, The State University of New York at Buffalo;

2. Department of Internal Medicine, Division of Allergic Diseases, Mayo Clinic Rochester, Minnesota

Understanding the Role of Fungi in the Pathophysiology and Treatment of Chronic Rhinosinusitis

68 © T O U C H B R I E F I N G S 2 0 0 7

Allergic Disorders

Niv Mor, BA, is a third-year medical student at The State University of New York at Buffalo, wherehe is pursuing an interest in otolaryngology. He graduated from Phi Beta Kappa with a BA inmathematics from Brandeis University in Waltham, Massachusetts. He studied the role of extra-cellular matrix proteins in smooth muscle relaxation of the lower esophageal sphincter at theHospital for Special Surgery in New York.

David A Sherris, MD, is Professor and Chair of the Department of Otolaryngology at The StateUniversity of New York at Buffalo, the Chief of Otolaryngology Services for the Kaleida HealthSystem, and a Director of the Gromo Institute and Sinus Center in Buffalo, New York. Hespecializes in rhinology and facial plastic surgery. He graduated from the University of Rochester School of Medicine in Rochester, New York, followed by a otolaryngology residency atthe University of Rochester Medical Center in Rochester. After a facial plastic fellowship at theUniversity of Washington in Seattle, he joined the Mayo Clinic Faculty in Rochester, Minnesota,and became the Chair of Facial Plastic Surgery.

Hirohito Kita, MD, is a Professor of Immunology and Medicine and the Director of the AllergicDisease Research Laboratory at the Mayo Clinic and Mayo College of Medicine. He specializes ineosinophil cell biology and airway inflammation. He graduated from the Mie University School ofMedicine, Japan. After a residency in pediatrics and a fellowship in pediatric allergy, he became apost-doctoral research fellow at the Mayo Clinic in Rochester, Minnesota.

Jens U Ponikau, MD is an Assistant Professor and Chair of Otolaryngology Research at The StateUniversity of New York at Buffalo, and a Director of the Gromo Institute and Sinus Center. After aresidency in otolaryngology he became a research fellow in otolaryngology and joined the MayoClinic faculty in Rochester, Minnesota. His research focuses on the role of fungi in the upper(chronic rhinosinusitis) and lower airways (asthma). He graduated from Medical School inHamburg, Germany.

E: [email protected]

Sherris_edit.qxp 10/10/07 15:22 Page 68


contain lymphocytes and antigen-presenting cells—were exposed to

different fungal antigens from species commonly found in the nasal and

paranasal sinuses. Ninety percent of CRS patients produced large amounts

of IL-5 in response to one particular fungus called Alternaria, while all CRS

patients produced large amounts of IL-13. This immune reaction was

specific for CRS since healthy controls did not react. In addition, IFN-gamma

(TH-1) production in response to Alternaria species was significantly

elevated compared with healthy controls.12

To further clarify the nature of the immune response to the fungi, serum

IgE and IgG antibodies to Alternaria were analyzed relative to the amount

of IL-5 production that was induced by Alternaria. Alternaria IgE levels did

not correlate with the level of IL-5 produced, confirming that this immune

response occurred independently of an IgE-mediated mechanism.

However, specific Alternaria IgG levels were strongly correlated with the

level of IL-5 produced. The production of IgG was significantly increased

in CRS patients compared with healthy controls, and the response was

independent of whether or not allergy was detected in the serum.13 Since

IgG levels are an indicator of the degree of exposure to antigen, this

indirectly suggests that the severity of this immune reaction is correlated

with exposure to Alternaria.

Thus, CRS patients, but not healthy controls, produce the cytokines

necessary to induce the eosinophilic inflammation, and certain

fungi—specifically Alternaria—trigger this reaction. Furthermore, this

pattern of cytokine production challenges the previously held belief that a

strictly inverse relationship exists between TH1 and TH2 immune responses.

Where Are the Fungi?

Given the role of fungal antigens in inciting this immune response in CRS

patients, previous studies that found negative fungal cultures and

negative fungal histopathology must be looked at in perspective.

Improved methods, such as collecting as much mucus as possible from

each specimen and removing the fungi from the mucus before incubating

the sample for culture, have increased the sensitivity of these tests.14

Results from these improved methods showed cultures positive for fungi

in 96% of patients with CRS; the healthy control group was also positive

for fungi (96–100%).14,15 These findings demonstrate that fungi are

present in the nasal and sinus cavity of all humans; however, only patients

with CRS seem to respond to them with cytokine production leading to

an eosinophilic inflammation.

In addition, the varying negative results from previous pathology

studies were due to the limitations of the Gomori methenamine silver

(GMS) stain used in these studies.16 GMS relies on the polysaccharide

present in the fungal cell wall to visualize the organism; however, the

fungi in CRS are present in the mucin, which is known to contain a

high concentration of polysaccharides. Chitin, a monosaccharide

derivative of glucose that is present in the cell wall of most fungi and not

present in mammals, appears to have greater sensitivity and specificity

for detecting fungal organisms (see Figure 1).17 More specifically, the

presence of Alternaria was further elucidated by more sensitive

Alternaria-specific methods such as polymerase chain reaction (PCR) or

antigen detection. These methods verified the presence of Alternaria

DNA or antigens in 100% of CRS patients.18 This is not surprising given

the fact that Alternaria alternata-specific antigens are found in 95–99%

of US households.19

What Do Eosinophils Do in the Sinuses?

Eosinophils were thought to infiltrate the sinus tissue of CRS patients and

accumulate below the basal membrane. Recently, however, it was

appreciated that they are actually only traveling through the tissue and

towards the mucus. On the luminal side of the epithelium the eosinophils

form in cell clusters, where they degranulate.20 Eosinophils in the tissue

were found largely intact. In contrast, the eosinophils in the luminal

clusters were releasing their cytotoxic eosinophilic protein, including

major basic protein (MBP), in concentrations that appear to far exceed

those necessary to induce epithelial damage (see Figure 2).20 Importantly,

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Figure 1: A Method to Identify Eosinophils in the Mucus

Panel A shows mucus from a patient with chronic rhinosinusitis that has been stained withGomori methenamine silver (GMS). No fungi can be visualized. Panel B is a serial section ofpanel A, which is stained with a fungal-specific chitinase immunofluorescence stain. Thepresence of the fungal elements (white arrows) can clearly be visualized in the mucus. The redarrows depict clusters of eosinophils in the mucus. Note that the fungal elements are localizedin the center of the eosinophilic clusters (GMS and Chitinase immunofluorescence staining;original magnification x400).

A. B.

Figure 2: Eosinophils Clusters in the Mucus

Panel A is a photomicrograph of mucus that is attached to tissue from a patient with chronicrhinosinusitis. The eosinophils form clusters (black arrows) in the mucus, and the epitheliumis eroded from the luminal side (yellow arrows). Panel B is a serial section of panel A. Theeosinophilic clusters are the site of diffuse major basic protein (MBP) release (white arrows).In contrast, the MBP in the tissue is confined within the intact eosinophils (red arrows). Thissuggests that the eosinophils in the tissue are in transit towards their final target in themucus. (Hematoxylin-and-Eosin and MBP immunofluorescence staining; originalmagnification x400).

A. B.

Given the great impact of chronic

rhinosinusitis, it is imperative to better

understand the mechanisms underlying

the disease process so that appropriate

treatment can be employed.


the epithelial damage is inflicted from the luminal side since the upper

layers of the epithelium are affected from the outside.8

Serial sections of the mucus using chitinase stains showed fungal

elements clearly localized in the center of crowded eosinophils. The

eosinophils form in clusters and surround the fungi in the same way

that they target and attack parasites (see Figure 1, red arrows and

Figure 3).17

The damage to the epithelium is explained by the high concentration

of eosinophilic toxic proteins, and the injured tissue presumably provides

an entry port for colonizing bacteria to invade the nasal mucosa of the

sinus cavity.5,8,21–25 In healthy control subjects, the absence of cytotoxic

MBP in the mucus explains the lack of epithelial damage and,

consequently, the lack of bacterial infections despite the presence of

bacteria. All of these findings explain why antibiotics, IgE-mediated

immunotherapy, and antihistamines have limited effectiveness as long-

term treatments of CRS.26,27

Does Alternaria Cause Eosinophils to Degranulate and

Release Its Toxic Proteins?

The observations that eosinophils target fungi in the mucus

and that the fungi are the sites of degranulation sparked further

investigations as to whether the fungi are actually causing the

eosinophils to degranulate. When seven fungi were tested (Aspergillus,

Penicillium, Alternaria, Candida, Curvularia, Bipolaris, and Cladosporium

spp.), only Alternaria and Penicillium induced a calcium-dependant

degranulation of eosinophils. In addition, only Alternaria caused

activation of eosinophils, as measured by the expression of CD63 and

CD11b, the production of IL-8, and an increase in intracellular calcium by

eosinophils. The specific activity was centered on an Alternaria antigen

with a molecular weight of 61kDa that is heat-labile and works through

a G-protein coupled receptor. In summary, Alternaria, but not other

fungi, caused activation and degranulation of eosinophils, whereas

neutrophils did not respond.13

Treatment of Chronic Rhinosinusitis

Glucocorticoids reduce inflammatory cytokines, inhibit the production,

migration and activation of eosinophils, and ultimately lead to eosinophilic

apoptosis.28 However, the administration of systemic glucocorticoids does

not address the trigger for the inflammation, and provides only temporary

relief with numerous undesirable side effects.29,30 Topical steroid sprays are

limited by constraints on delivery into occluded nasal and paranasal cavities.

However, they recently demonstrated efficacy in patients with nasal polyps,

where they reduced the nasal obstruction and the size of the polyps in two

independent trials.31,32

Another therapeutic target would be to reduce the antigenic stimulus in the

sinus and nasal cavities that activates the inflammatory response. Due to the

extra-mucosal, non-invasive nature of the fungi, systemic antifungals would

have to be given at a high dose in order to reach effective concentrations in

the mucus. In view of the potential risk of renal, liver, and cardiac toxicity of

a long-term treatment, more safety and efficacy data are necessary to justify

broad use of systemic antifungals.

Recent studies have tested the effects of direct administration of

antifungal agents. Intranasal treatment with amphotericin B reduced

both the inflammatory mucosal thickening (as seen by computed

tomography (CT) scan) and improved the disease stage (as seen by

Allergic Disorders

70 U S R E S P I R A T O R Y D I S E A S E 2 0 0 7

Figure 3: Eosinophils Forming Around a Fungal Element

The above image is an transmission electron micrograph of eosinophils (black arrows) formingaround a fungal element (red arrow). Note the intimate relationship of the eosinophils engulfingthe fungi in preparation to release their toxic major basic protein (yellow arrow).

Figure 4: Before and After Treatment of Chronic Rhinosinusitis

Coronal computed tomography scans of a patient with chronic rhinosinusitis before and after treatment with intranasal amphotericin B. Panel A shows inflammatory mucosalthickening partially occluding the nasal cavity and the paranasal sinuses, which is significantly reduced in panel B after intranasal treatment with amphotericin B. The challengeof intranasal delivery is further illustrated by residual disease in the hard to reach upperethmoid sinuses (*).

A. B.

The administration of systemic

glucocorticoids does not address the

trigger for the inflammation, and

provides only temporary relief with

numerous undesirable side effects.


endoscopy) (see Figure 4).26,27 In addition, patients receiving amphotericin

B had reduced levels of eosinophil-derived neurotoxin (EDN)—an

intranasal marker of eosinophilic inflammation—compared with patients

in the placebo group. Interestingly, the daily intranasal administration of

the placebo solution, containing colored sterile water, worsened the

inflammation when seen by CT scans, did not improve the endoscopic

staging, and increased levels of EDN.26 These results suggest that the

therapeutic benefit of an intranasal amphotericin B solution is not due to

the mechanical cleansing of the paranasal sinuses, but due to a

therapeutic effect of the amphotericin B.

Nasal polyposis is considered to be the late and ultimate stage of CRS

that presents with edematous swellings in the sinus tissue. Treatment

with nasal lavage of amphotericin B resulted in the total disappearance

of the polypoid mucosa.33,34 The efficacy of antifungal treatment on nasal

polyps was related to the severity of disease: 62% of patients in stage I

polyposis and 42% of patients in stage II were cured using this

treatment. None of the patients in stage III were cured, presumably due

to reduced delivery of the topical medication in severely obstructed

patients.33,34 Previously, lysine acetylsalicylate (LAS) was used to treat nasal

polyps due to its antiproliferative effects on fibroblasts and its ability to

reduce the recurrence of nasal polyps more effectively than surgery.

However, when patients were treated with a combination therapy of

intranasal LAS and amphotericin B, they had significantly lower rates of

recurrence of nasal polyps than those patients who were treated with

LAS alone.35

Conclusion

Alternaria, a ubiquitous airborne mold, is present in CRS patients

as well as in healthy controls. However, only the immune cells of CRS

patients react to the presence of Alternaria with the production of

cytokines crucial for inciting the eosinophilic inflammation seen in this

disease. This cytokine-driven immune response occurs independent of IgE

production. In addition, Alternaria induces the degranulation of

eosinophils and the release of their toxic protein. In CRS patients one can

appreciate the migration, cluster formation, degranulation, and targeting

of extramucosal fungi by the eosinophils. Antifungal therapy shows

promise in reducing the trigger for the eosinophilic inflammation, and

consequently the inflammatory mucosal thickening. However, drug

delivery, dosage, and frequency and duration of treatment must be

further optimized. ■

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Antifungal therapy shows promise

in reducing the trigger for the

eosinophilic inflammation, and

consequently the inflammatory

mucosal thickening.


71U S R E S P I R A T O R Y D I S E A S E 2 0 0 7
