Inflammation of the nasal cavity and the contiguous paranasal sinus mucosa is termed chronic rhinosinusitis (CRS). CRS symptoms last for more than 12 weeks and may or may not be associated with nasal polyposis (NP), referred to as CRSwNP and CRSsNP, respectively [1]. NP, which is associated with inflammation of the nasal cavity and paranasal sinuses with persistent nasal discharge, anosmia, stuffy nose, and headache, is a devastating illness of unknown etiology [2]. The two forms of CRS have different underlying pathophysiological mechanisms, response to treatment modalities, and prognosis. NP forms an important social issue with high global incidence (2–5%) [3] and unsatisfactory treatment outcomes [4]. CRSwNP is further categorized as eosinophilic and non-eosinophilic based on the nasal lavage cytology pattern [5]. Eosinophils have been implicated in the formation of edematous polyps from sinuses to nasal cavity [1].

Several bacteria and fungi can cause CRS [3]. Fungi, especially Aspergillus flavus can cause severe acute or chronic sinusitis in immuno-competent hosts. In response to airborne fungi entrapped in the sinonasal mucus, the toxic granules released from eosinophils and mucosal inflammation cause CRS. Inflammatory cytokines play a role in controlling the fungal burden and reducing inflammation [6^,7].

The pathophysiology of polyp formation through the recruitment and activation of inflammatory cells and their survival involve cytokines. Though both Th1- and Th2-type cytokines are involved, their roles are not yet fully understood. Coa et al [8] reported high expression of Th1, Th2, and Th17 in CRSwNP patients than in controls. Zhang et al [9^,10] observed a non-eosinophilic Th1/Th17-biased response in patients with CRSwNP in an Asian population. Th17 cells, which release the pro-inflammatory cytokine IL-17; and T regulatory (Treg) cells, which secrete the anti-inflammatory cytokine IL-10 and transforming growth factor-β (TGF-β), are distinct from Th1 and Th2 T-cell subsets. Responding Th17 cells are considered vital in autoimmunity, inflammation, and allergic reactions [11^,12]. Moreover, IL-2 and TGF-β act as growth factors for Treg cells and promote their differentiation, while TGF-β along with IL-6 promotes the differentiation of IL-17 cells [13]. The inflammatory cells that infiltrate nasal polyps differ in Asian and Western patients; this is attributed to the different genetic predispositions and environmental conditions. Hence, an attempt to understand the underlying pathogenesis in an Indian population was undertaken. We investigated the serum levels of various cytokines (IL-1β, IL-2, IL-4, IL-6, IL-17, IL-21, IL-27, TGF-β) and immunoglobulin E (IgE) in CRSwNP patients with and without A. flavus infection and compared them with the levels in healthy controls. We attempted to correlate the findings to understand the underlying immunopathogenesis of the CRSwNP.

Various potential etiological factors, such as anatomical variants, microbial and, particularly, fungal infection and/or colonization, atopic response, intolerance to acetyl salicylic acid, and sometimes combinations of all these, have been implicated in the CRS process [19^,20]. The upregulation of inflammation of the nose, laterally, by these factors results in NP development [2]. The pathogenic process is known to begin as a result of dysregulation of the Th1/Th2 balance with excessive Th1 or Th2 activation. IL-17 has recently been implicated in regulating NP inflammation by attracting eosinophils and subsequent tissue reaction. Shen et al [21] have described atopy as an important factor in NP pathogenesis that may expand the disproportionation of Th17/Treg balance, thereby aggravating polyposis via the IL-17 inflammatory pathway. Additionally, CRSwNP is associated with high eosinophil levels, which distinguishes it from CRSsNP; however, in early stages of polyposis, the levels of IgE may not differ considerably from those in non-polypoid sinusitis [1].

Based on histopathological criteria, 41.93% of cases were categorized as allergic fungal rhinosinusitis, characterized by a fungal allergen-mediated IgE allergic response and associated with abundant accumulation of eosinophils in the mucin as described by Bent and Kuhn [22]. High total IgE can induce an allergic inflammatory cascade, involving the recruitment of eosinophils and basophils, leading to a series of cellular events [23]. Further, high specific IgE affects the underlying pathogenesis, leading to adverse prognosis and poor management of patients.

Fungal allergens have been shown to elicit IgE-mediated allergic and possibly, type III (immune complex)-mediated mucosal inflammation in atopic hosts [24]. In such sensitized individuals, fungi persist locally and stimulate destructive immune responses. Further, eosinophilic buildup in the mucin boosts inflammatory mediators in the extended sinuses. The association of high IL-1β and TGF-β with low IL-27, IL-6, and IL-2 indicate their involvement in Th17 subset differentiation in CRSwNP patients with A. flavus infection, suggesting Th17/Treg equilibrium as a key factor in modulating the immune responses and is perhaps crucial for disease progression in these patients [24]. Our findings are similar to previous findings in a Chinese population, which suggested that a skewed Th17 response with impaired Treg function is crucial both locally and systematically for NP pathogenesis [21]. However, the comparative IL-2 increase in CRSwNP patients without A. flavus infection may have suppressed the levels of IL-17, IL-1β, TGF-β, and associated cytokines.

It is important to understand that cytokines act sequentially, in concert or in conflict, in a given immune response, depending on whether the host–pathogen interaction is dominated by the conidial or the hyphal form of a fungus [25]. Repeated exposure to Aspergillus conidia induces the co-evolution of Th1, Th2, and Th17 responses in the nasal mucosa of patients with CRS. Low IL-2 level is a poor trigger for Treg cells and favors the predominance of Th17 subsets. The absence of cytokines IL-6 and IL-27 does not hinder Th cell differentiation mediated by IL-21 and IL-17 [23]. Our study showed that the low levels of IL-6 and IL-27 and high level of IL-1β might be responsible for Th17 cell proliferation; however, the role of IL-21 in inducing the production of IL-17 from naive T cells cannot be ignored [26]. Therefore, the differentiation of Th17 cells might involve the cooperation of IL-6 and/or IL-21 with TGF-β. This is supported by the study of Korn et al [26], which suggested that cooperation of IL-21, a member of the IL-2 cytokine family, with TGF-β induces the generation of Th17 cells from naïve IL-6-deficient T cells, and that T cells deficient in IL-21 receptor generate a defective Th17 response. We also observed a lethargic Th1 response, which counter balances persistently high IL-17 levels in patients with CRSwNP with A. flavus infection, leading to tissue damage. The low IL-4 level as indicated by our study correlates well with the suppressed Th2 response in CRSwNP as a whole.

Persistent fungal exposure highlights a role for IL-17 in upholding the inflammatory response in a majority of patients with CRSwNP with A. flavus infection. In our study, the predominant Th17/Th1 response favored an ongoing inflammatory process in patients with average disease duration of 8–10 months that abrogated the Th2 response. Hence, it is important to identify the sustained existence of an antigenic stimulus at the infection site, which provides a continuous inflammatory stimulus for developing a CD4-positive T-cell response. IgE level, eosinophils in the local tissue/biopsy, and peripheral eosinophilia are all hallmarks of a Th2 response, while low IL-4 and IL-6 are indicative of a dose-dependent adaptive response that has been overtaken by a chronic inflammatory response under the influence of IL-17 [27].

In the absence of a strong Th1 response, Tregs facilitate an antigen-specific Th17-mediated response in the presence of various cytokines and inhibit fungal clearance. Because Tregs ultimately have a role in fungal clearance, their expansion is necessary to limit the disease. Like Treg cells, proliferation of the Th17 response is stimulated by fungal cell-wall components via dectin-1 interaction [28]. Additionally, Th17 differentiation can be facilitated by Tregs, and Tregs themselves can convert to Th17 cells [29] in a process assisted by dendritic cells [30]. This may drive the initial Th17 response aided by the diminished Th1 response, since Th1 and Th2 cytokines inhibit Th17 development. Murdock et al [27] observed that Th17 cell levels increased and acquire a dominant adaptive response on repeated exposure to A. fumigatus conidia, while the Th2 cell levels remained stable or were reduced. The time-dependency of IL-17 function and its role in exacerbating and/or diminishing Th2 inflammatory responses have been shown in several studies. Thus, as an alternative immunological pathway to repeated low fungal allergen exposures, a Th17 response might arise.

In conclusion, the role of IL-17 in driving CRSwNP in A. flavus-positive patients, with either eosinophilic or neutrophilic inflammation promoted by high TGF-β, was revealed. However, whether the role of cytokines in instructing CD4-positive T-cell differentiation is fungi-specific or not needs to be elucidated for conclusively identifying IL-17 as an important marker for disease progression. Whether the role of raised IgE in causing mast-cell activation and inflammation is independent of the type of stimulus or allergen, which requires further investigation, raises concern regarding the effective, appropriate, and targeted therapeutic intervention in allergic disorders like CRS and associated relapses.

The authors declare no conflict of interest.

This work was financially supported by the grant from the Department of Biotechnology (DBT), Government of India, New Delhi [No. BT/PR5191/MED/29/447/2012].

Serum levels of various cytokines in healthy controls and patients with chronic rhinosinusitis with nasal polyposis (CRSwNP) and A. flavus infection. The bars in (A) show the levels of downregulated cytokines (IL-2, IL-4, IL-6, and IL-27), and those in (B) show the levels of upregulated cytokines (IL-17, IL-21, IL-1β, and TGF-β), compared with healthy controls. ^*P<0.05; ^**P<0.01; ^***P<0.001.

Serum IgE levels in healthy controls and patients with chronic rhinosinusitis with nasal polyposis (CRSwNP) and A. flavus infection.