Although chronic rhinosinusitis (CRS) is a common chronic health condition, affecting 10–15% of the European and US population in recent epidemiologic studies (1, 2), its underlying pathogenetic mechanisms remain unclear. CRS probably represents a heterogenous group of diseases resulting from a multifaceted interaction between the host and the environment. The microorganisms colonizing the airways have been identified as sources of signals for the innate, as well as adaptive, mucosal immune response. Variations such as skewing of T-cell populations and polarized cytokine patterns may modulate an abnormal response to the presence of environmental triggers within the upper airway (3, 4). Research into the staphylococcal superantigens elegantly reflects the interplay between microorganisms and the local immune system, and their role as disease modifiers in nasal polyp disease (CRSwNP) is now well established (4). Biofilms are a relatively new concept in the CRS literature, but do have several features that might be relevant when considering the impact of bacteria such as Staphylococci (5).
CRSwNP and CRSsNP (without nasal polyps) are increasingly recognized as distinct disease entities based on cytokine, mediator, and cellular profiles (6). CRSsNP is largely a fibrotic, remodeling disease driven by T-helper1 (Th1) cytokines such as IFN-γ with normal T-regulatory cell function (7, 8). Conversely, polyp formation of the mucosa in CRSwNP ensues following escape from the inhibitory function of T-regulatory cells (evidenced by a reduction in FoxP3 and TGF-β), enabling the T-helper2 (Th2) cells to predominate and their cytokines, in particular IL-5, to recruit and activate eosinophils (9). The presence of superantigens has been consistently demonstrated in 20–50% of Caucasian patients with CRSwNP, but rarely in CRSsNP or control subjects. The Th2-biased cytokine patterns are further exaggerated in this subgroup of CRSwNP, linking comorbid asthma to nasal polyposis (10, 11). However, the genesis of the eosinophilic and occasional neutrophilic responses in the remaining nasal polyposis patients is still elusive, underlining both the heterogeneity of this condition and the limitations of our current knowledge.
A biofilm is defined as a community of bacteria that are encased in an exopolysaccharide matrix they have produced and are irreversibly attached to a surface (12). Biofilm bacteria exhibit unique characteristics with respect to both growth and metabolism. Resulting biofilm-mediated diseases share common features, being chronic diseases with repeated acute exacerbations, variable culture rates, and extreme antibiotic resistance. These are characteristics that are commonly seen in our CRS population; hence, the biofilm hypothesis has recently been applied to CRS. The existence of biofilms in CRS has now been well established (13–16). Recent work from our department has outlined the polymicrobial nature of CRS biofilms (17), with Staphylococcus aureus and H. influenzae featuring prominently (17–19). The presence of biofilms has been associated with poor evolution following sinus surgery (20, 21); S. aureus biofilms are linked to more severe and surgically recalcitrant disease, whereas H. influenzae biofilms are generally seen in mild disease that is highly responsive to current management paradigms (22).
A direct role for biofilms in CRS disease initiation remains circumstantial. By evaluating both the cytokine patterns associated with staphylococcal biofilms and their coexistence with superantigen-specific IgE within the sinuses, we hope to provide insights into the adaptive immune responses that characterize S. aureus biofilm–associated CRS and possibly further clarify their pathogenic role in this disease.
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The results of this study not only confirm previous findings of a skewed cytokine profile in CRS patients with nasal polyposis and the presence and impact of staphylococcal superantigens on the mucosal inflammation but also demonstrate a polarized immune response in the presence of S. aureus biofilms. Despite circumstantial evidence, little published research has specifically examined the immune consequences of biofilms in CRS. Our discovery of an association between S. aureus biofilms and an eosinophilic, Th2-polarized inflammation in CRS, irrespective of polyp status and independent of the superantigen pathway, implies a direct link between microorganism and host. This may finally allow definitive conclusions on the pathogenic role of biofilms in this poorly understood disease.
The role of S. aureus in CRS is expanding as its importance as a pathogen is increasingly recognized. Superantigens released by S. aureus have a well-defined role in the pathogenesis of CRS, acting as disease modifiers in the CRSwNP phenotypic subgroup, a fact confirmed for an Australian population by the current results (26). S. aureus is known to be able to reside intracellularly and intramucosally in the sinonasal mucosa (27–29), and nasal colonization rates with S. aureus exceed 60% in the CRSwNP subgroup (11). These are both potential reservoirs for superantigen release in the sinuses. Alternatively, bacteria existing in the biofilm form might act as a nidus for planktonic bacteria dispersing into the mucosa, a theory that was confirmed in the current study.
Methodologically, we utilized FISH because of its ability to identify individual biofilm-forming species (24). S. aureus is the most common biofilm-forming organism in the CRS population (17), and its presence is a predictor of more severe disease (22). In contrast, H. influenzae biofilms are associated with a favorable disease course (22). Our data on H. influenzae biofilm did not demonstrate an impact on the mucosal response, and this confirmed the clinical observation.
The results of this investigation associate the presence of S. aureus biofilms in polyp patients skewing toward the T-helper2 pathway with a resultant eosinophilic inflammatory milieu. This may occur both dependent and independent of the superantigen pathway. The statistically significant association between biofilms and superantigens suggests that S. aureus biofilms may act as a nidus from which planktonic S. aureus and superantigens are released into the paranasal sinuses. Given the combined presence of S. aureus biofilms and superantigen-specific IgE was only observed in 12/26 CRSwNP patients, other mechanisms must be at play if S. aureus biofilms do indeed interact with the host in CRS. To differentiate the effects of S. aureus biofilms and superantigens, a number of different analyses were carried out. Firstly, when the non-superantigen CRS patients (both with and without polyps) were analyzed, IL-5, IL-6, and ECP were all significantly elevated in the biofilm-positive group. Thus, the release of superantigens is not required for a skewing of the T-helper2 host response in the presence of S. aureus biofilms. We were also able to replicate similar findings in both the CRSwNP and CRSsNP subgroups when analyzed separately. Within the CRSwNP subgroup, the S. aureus biofilm group has higher IL-5 and ECP, suggesting a further delineation of what is already known to be an eosinophilic disease. Interestingly, within the CRSsNP subgroup, the biofilm-positive patients are associated with moderately higher T-helper2 cytokines IL-5 and IL-6 but not a raised ECP. CRSsNP is generally thought not to be eosinophilic in nature, and this may in part explain this result. Alternatively, this may be a type 2 statistical error owing to insufficient numbers. Only further patient recruitment and adaptive immune evaluation will answer this.
Perhaps most importantly though, in attempting to differentiate the effects of S. aureus biofilms from staphylococcal superantigens, is the results of the linear discriminant analysis (Fig. 3). A linear discriminant analysis allows the variation in the nine cytokines to be statistically reduced into two components to permit visualization on a biplot graph in which each cytokine is represented by a vector. In Fig. 3, the T-helper2 cytokines trend toward the left and downwards. The T-helper1 cytokines are toward the right. Importantly, the effect of S. aureus biofilms, independent of superantigen release, can be differentiated on the basis of how much they contribute to the vectors of IL-5, IL-6, TGF-β1, and particularly ECP. Patients with S. aureus biofilms but without superantigens (group 3) shares a stronger relationship with all three of these cytokines than does the superantigen IgE–positive, biofilm-negative patients (group 2). This implies that the link between S. aureus biofilms and both the T-helper2 pathway and an eosinophilic inflammatory response is independent of the effect of superantigens, reinforcing the results of the subgroup analysis discussed above which first suggested this independent association. Conversely, the presence of superantigens alone correlates with IgE levels, and the presence of both superantigens and S. aureus biofilms (group 1) produces an amplified T-helper2 response in CRSwNP, perhaps advancing our understanding of the pathomechanics of the CRSwNP subgroup of this disease. This is important, new information because despite the clear evidence of eosinophilic inflammation in CRSwNP, not all of these patients have detectable superantigen IgE. There must be another mechanism, and S. aureus biofilms may be one of the answers. The link between the innate and adaptive immune response appears to be important, yet not understood in CRS. The data presented in our comparison trees (Fig. 4) confirm the hypothesis that the mode of action of superantigens and S. aureus biofilms may be different. Elevated total IgE and MPO distinguish the presence of superantigen IgE, whereas the presence of S. aureus biofilms associates with elevated ECP and IL-5 in particular, in addition to TGF-β1. The combined elevation of both ECP and IL-5 is 100% predictive of S. aureus biofilm presence, whereas the combined elevation of total IgE and MPO is 100% predictive of superantigen-specific IgE in the sinonasal mucosa. Thus, the classification tree analysis supports the discriminant analysis in distinguishing the effects of Staphylococcus aureus biofilms and superantigen.
A link between biofilms and T-helper2 polarization has been found before. Chronic periodontitis is a classical biofilm-mediated disease in which their role has been extensively researched over the last 15 years. Porphyromonas gingivalis is associated with an exuberant T-helper2 response that subsequently dictates disease initiation and progression (30, 31). P. gingivalis is thought to incite a poor innate immune response leading to polyclonal B-cell activation and a T-helper2 response. Ultimately, nonprotective antibodies are produced and a chronic infection established. In contrast, Aggregatibacter actinomycetemcomitans and Prevotella intermedia are biofilm-forming organisms that do not associate with a T-helper2 response (32). These observations seem plausible at another host–environment interface, namely the sinonasal mucosa, where the differential pathogenicity of S. aureus and H. influenzae may influence disease characteristics. It is interesting to note that the only other paper in the literature to examine cytokine profile of biofilm-positive CRS patients had contrastingly different results. In a study of 19 CRS patients, the presence of biofilms on the sinus mucosa was associated with a skewing toward the T-helper1 pathway. In contrast to the present study, however, the biofilm-forming species were not determined by Hekiert et al. (33).
Despite the evidence produced by this study, a number of questions remain. The interrelation between the T-helper2-biased mucosal immune response and S. aureus biofilm formation needs to be clarified. Profiting from a pre-existing T-helper2 bias within the mucosa, S. aureus may switch to the biofilm phenotype and establish itself within the damaged mucosa. Alternatively, S. aureus biofilms may induce the T-helper2 bias, which initiates the specific mucosal changes already described. Host lactoferrin is reduced in biofilm-associated CRS (34), and alternatively activated M2 macrophages display deficient microbicidal activity against a range of microorganisms, including S. aureus (35), associated with bacterial persistence (36). Recent data suggest an increase in M2 macrophages in CRSwNP with reduced phagocytosis and killing abilities (37). The sequence of these events still needs further study.