ANEXOS

The separation of the innate and the adaptive immune respon-ses into distinct compartments is artificial; in vivo, these two arms of the immune response are mutually supportive and distinctions have been blurred. Nevertheless, the separation is useful for mechanistic and temporal explanations of immune defence across mucosal barriers. Specifically, if penetration of the mechanical and innate defences is sufficiently strong, an adaptive immune response will be initiated and maintained;

the overall response will be specialized to address both the specific nature and the strength of the insult. Cytokine crosstalk between epithelial cells (ECs), innate lymphocytes (ILCs), and dendritic cells (DCs) plays a key role in matching the appropriate adaptive response to foreign stimuli. In health, this maintains mucosal homeostasis with 1) tolerance of potential allergens and commensals and 2) defence against pathogens without the development of chronic inflammation. Adaptive immunity is typically divided into a T-cell response and a B-cell response, and these will be discussed as they relate to CRS.

Antigen presentation, dendritic cells and T cell activation The initiation of T cell responses requires that naïve T cells re-cognize antigens presented by dendritic cells under conditions that require multiple receptors and co-stimulatory molecules.

Antigen presentation is a transition phase from the innate to the adaptive response and is mediated mainly by DCs⁽²⁷⁴⁾. DCs can activate both innate and adaptive immunity via antigen capture, presentation of antigen to immature T cells, and secretion of soluble inflammatory mediators. DCs are influenced by the col-lective cytokine response from ECs and ILCs, and this is believed to be pivotal in shaping T-cell differentiation⁽²⁷⁷⁾. Plasmacytoid dendritic cells are decreased in patients with a more severe type 2 profile, suggesting an important role of the cytokines milieu in their functional response or that plasmacytoid dendritic cell could mitigate the inflamed process found in polypoid tissue^(349,

350). Zheng et al. showed increased expressions of IL-17RB and ST2 on myeloid DCs associated with enhanced local type 2 inflammation in polyp⁽³⁵¹⁾. Shi et al. detected distinct subsets of lesional DCs in eosinophilic and noneosinophilic CRSwNP, which could prime Th2 cells, and Th1/Th17 cells respectively in these endotypes⁽³⁵²⁾. Lin et al. investigated the functional status of dendritic cells (DCs) in polyp/mucosa from 30 CRSwNP and 10 control subjects and detected infiltration of DCs in NP, with the majority being mature DCs, and that DCs are able to interact with T cells via the CD40/CD40L co-stimulatory factor⁽³⁵³⁾. Antigen presentation is regulated by co-stimulatory molecules.

Programmed cell death-1 (PD-1) is a negative regulator of T-cell responses⁽³⁵⁴⁾. Kortekaas Krohn et al. measured expression of PD-1 pathway molecules by RT-qPCR on tissue from 21 CRSwNP

and 21 control subjects and detected higher PD-1 expression in CRSwNP compared to controls independently of atopic

sta-tus⁽³⁵⁴⁾. In accordance with this, high PD-L1 levels were

associa-ted with Asian eosinophilic CRSwNP compared to controls⁽³⁵⁵⁾. Overall however, at this point, only limited evidence exists to support the hypothesis that defects in antigen presentation are significant in CRS pathogenesis.

T cells

T cells have multiple roles in immune defence including defence against intracellular and extracellular infection⁽²⁷⁴⁾. T cells acti-vate and recruit effector cells including phagocytes, kill infected cells and provide help for B cells in immunoglobulin production.

Memory T cells are active as a durable form of innate activity at barrier surfaces such as the sinus mucosa.

A major role of CD4+ T helper cells is to send signals to other types of immune cells, including CD8+ killer cells^{(274, 356)}. Loss of T-helper cell function leaves the body vulnerable to a wide range of infections as have been detected in untreated HIV infection⁽²⁷⁴⁾. CD4+ T cells may differentiate into different subsets of effector cells that produce distinct sets of cytokines that as-sist in defence against various types of microbial infections in tissues, and a fourth subset that activates B cells in secondary lymphoid organs: Th1, Th2, Th17 and follicular helper T cell⁽²⁷⁴⁾. A fifth T subset are T regulatory (Treg) cells that help turn the process off. Activated CD4+ cells produce a mixture of the cha-racteristic cytokines⁽²⁷⁴⁾. The Th1 subset is induced, with the help of ILC1s, by microbes that are ingested by phagocytes (primarily macrophages)⁽³⁵⁶⁾. Th1 cells stimulate phagocyte-mediated killing of ingested microbes via secretion of interferon-γ (IFN-γ) and tumor necrosis factor -α and –β (TNF-α and TNF–β), leading to macrophage activation and enhanced antigen presentation, B-cell help and class switching to production of IgG subclasses and local tissue inflammation and neutrophil activation^{(356, 357)}. Th2 cells are induced by parasitic worm infections and promote IgE-, mast cell-, and eosinophil-mediated destruction of these parasites with the help of ILC2 cells^{(274, 358)}. Th2 cells secrete the Type 2-cytokines IL-4, IL-5, and IL-13, which have important protective effects that include eosinophil effector mechanisms, immunoglobulin class switching to IgE and IgG4 and mucus production⁽³⁵⁹⁾. The Type2-cytokines also stimulate the alter-native pathway of macrophage activation and shut down the classical pathway mentioned above. This terminates prolonged and potentially damaging reactions. Alternative M2 macropha-ges secrete growth factors that act on fibroblasts to increase collagen synthesis and induce fibrosis fostering tissue repair.

Th17 cells develop in response to extracellular bacterial and fungal infections and induce inflammatory reactions destroying these organisms. Th17 stimulate recruitment of neutrophils and monocytes and the production of defensins via secretion of

IL-17A, IL-17F, and IL-22.

CD4+ T cells differentiate and acquire distinct functions to com-bat specific pathogens but can also adapt their functions in res-ponse to changing circumstances^{(358, 360)}. Although this phenoty-pic plasticity can be potentially deleterious, driving pathology, the T-cell flexibility also provides important benefits that have led to its evolutionary preservation⁽³⁶⁰⁾. Studies of the human T cell response to microbes have advanced our understanding of CD4+ T cell functional heterogeneity, in particular with the discovery of a distinct Th1 subset involved in the response to Mycobacteria and the characterization of two types of Th17 cells specific for extracellular bacteria or fungi⁽³⁵⁶⁾.

In CRS, the protective adaptive pathways go awry, usually with an excessive and prolonged response⁽³⁶¹⁾. In support of this, the numbers of regulatory T cells (Tregs) are generally reduced in CRS and this has been interpreted as diminished Treg activity leading to chronicity⁽³⁶²⁾. For the other Th subsets however, elevated numbers have been associated with CRS⁽³⁶³⁾. Elevated numbers of Th2 cells and eosinophils have been reported with Western CRSwNP, while increased Th1/Th17 and neutrophilia are associated with Asian CRSwNP and CF polyps(343, 344, 363). There is emerging evidence to suggest that the prevalence of Type 2 CRS is increasing in Asia however⁽³²²⁾. The aetiologic factors underlying this shift are both unknown and likely complex.

Nevertheless, the speed of the process suggests changes in environmental exposures with secondary epigenetic variation as a root cause. Importantly, an overall increase in T2 diseases was observed in Western countries beginning in the early 20th cen-tury, and the changes in Asia appear to be manifesting in those areas adopting a more Western life style. Further study of these real-time changes in Asian CRS may be useful in identifying key environmental determinants that, with avoidance, could lead to a reduction in the incidence of CRS.

CD4+ T cells that express Foxp3 and with immune suppres-sive activity are known as CD4+Tregs⁽²⁷⁴⁾. Tregs develop in the thymus or peripheral tissues on recognition of self-antigens and suppress the activation of potentially harmful lymphocytes specific for these self-antigens. They are also essential for main-taining tolerance and immune homeostasis^(364-369). As mentioned above, Tregs appear to be reduced in CRS and autoantibodies have been reported. Studies show that multiple Th subsets may be activated in the same CRS patient, that the cytokines produ-ced may suppress or augment the other subsets, and this may be part of normal immunoresponse, or may affect development or clinical presentation of CRS, or may affect treatment course^(342,

370).

CD8+ cells activated by antigen and other signals differentiate into cytotoxic T cells (CTL, T-killer cell) that are able to kill infec-ted (particularly with viruses) cells expressing the antigen⁽²⁷⁴⁾. CTLs also kill malignant and other damaged cells⁽²⁷⁴⁾. Memory T cells recirculate from the tissues to the peripheral blood. As

mentioned above, T cell levels are higher in CRS but the CD8+/

CD4+ T cell ratio is also higher, indicating particular enhance-ment of the cytotoxic T cell population^{(371, 372)}. Any specific role for cytotoxic T cells in CRS pathogenesis, however, remains unclear⁽³⁷²⁾.

Cell adhesion molecules located on the cell surface are involved in binding with other cells or with the extracellular matrix.

Adhesion molecules on leukocytes, fibroblasts and vascular endothelium, enabling leukocyte extravasation from vessels and migration to sites of inflammation⁽³⁷³⁾. Increased expression of intercellular adhesion molecule-1 (ICAM-1) in activated eosinop-hils is associated with eosinophil extravasation and infiltration into NP tissue, and seems to initiate also mucosal remodeling of

NP⁽³⁷⁴⁾. Fibroblasts´ expression of vascular cell adhesion molecule

(VCAM) has been shown to attract eosinophils and mast cells contributing to Type 2 cytokine skewing, while ICAM attracts neutrophils and to a lesser degree, eosinophils⁽³⁷⁵⁾. VCAM expres-sion was lowest in controls, higher in CRSsNP, and highest in CRSwNP, while ICAM expression was elevated in both CRS sub-types⁽³⁷⁵⁾. The L-selectin ligand, peripheral lymph node addressin (PNAd), was preferentially induced in the nasal vasculature of eosinophilic CRS facilitating tissue recruitment from blood⁽³⁷⁶⁾. It is not clear if primary defects in cell adhesion markers in parti-cular or effector recruitment in general, play any key role in CRS pathogenesis. Specifically, the above findings may simply be the predicted secondary effect of the tissue inflammation present in the sinus mucosa of the CRS patient. Regardless, this may lead to innovative treatments via limiting the influx of eosinophils and neutrophils into the sinus tissues.

Humoral immunity is the type of host defence mediated by anti-bodies, which are produced and secreted by B cells and plasma cells, functioning both in circulation and in peripheral tissues such as sinonasal mucosa⁽²⁷⁴⁾. Antibodies use their antigen bin-ding regions (Fab) to bind and block harmful effects of microbes and toxins. Heavy-chain isotype (class) switching and affinity maturation enhance the protective functions of antibodies⁽²⁷⁴⁾. The major functions of antibodies are to block or neutralize the infectivity of microbes, coat microbes and promote their ingestion by NK-cells, to activate (IgE) mast cell and eosinophil mediated reactions against helminthic parasites, and activation of the complement system vs. Physiologically tonic secretion of secretory IgA works in concert with other innate protective fac-tors and mucociliary flow to limit mucosal colonization without tissue-damaging inflammation⁽³⁷⁷⁾. This IgA is of low affinity and generated by a T cell independent process by extrafollicular B cells. In the case of an active breach of the respiratory mucosa, higher-affinity IgA secretion may increase and, in combination with help from IgG, a robust inflammatory response ensues⁽³⁷⁸⁾. These are high affinity, T dependent immunoglobulins produced by follicular B cells and plasma cells. IgG is the most common circulating antibody with four subclasses (IgG1-4) and a half-life

of 7-21 days. IgG has several effector functions: it neutralizes microbes and toxins; opsonizes antigens; activates the classical pathway of the complement system; is responsible for NK cell cytotoxicity events and feedback inhibition of B cells⁽²⁷⁴⁾. IgD is an enigmatic antibody isotype best known when co-expressed with IgM on naive B cells. Class switching to IgE is regulated by Type 2 cytokines and this immunoglobulin plays several important physiologic roles that include antigen presentation, increased mast cell survival, mucosal homeostasis, and defence against viruses, bacteria, fungi and parasites(312, 348, 379-383). The Fc portions of IgE and IgA are often mounted on mast cells and eosinophils, respectively⁽²⁷⁴⁾. The antigen selective portions can bind parasites, sterically inhibiting their ability to invade and also leading to degranulation, which may trigger inflammation and some degree of tissue damage.

B cells and immunoglobulins have been implicated in CRS and antibody defects are the most common immunodeficiencies, typically associated with CRSsNP⁽³⁸⁴⁾. Low immunoglobulin pro-tection can lead to recurrent acute sinus infection and ultimately CRSsNP⁽³⁸⁵⁾. Most cases of CRSsNP do not have an immunoglo-bulin deficit however, and in fact, a subset of CRSsNP patients has high tissue levels of IgD, although any role in pathogenesis remains uncertain.

B cell activation with excessive local antibody production has more commonly been associated with CRSwNP(369-371, 386). Polyp tissue from CRSwNP patients has been found to contain high levels of B cells, plasma cells, follicles that resemble germinal centres, and high levels of IgA, IgM, IgG and IgE that indicate local production of immunoglobulins(337, 369-372, 387, 388). Antibody production in CRSwNP is driven in part by elevated levels of the BAFF cytokine (B-cell activating factor) in nasal polyp tissue⁽³⁸⁹⁾. In addition to B-cell proliferation, BAFF has also been associ-ated with isotype class switching to IgE and IgA as well as the development of autoimmune immunoglobulins. Tan et al. was the first to demonstrate this, with anti-dsDNA IgG and IgA auto-antibodies at increased levels in nasal polyps in comparison with controls, suggesting a role for autoimmunity in the more seve-rely affected CRSwNP patients⁽³⁴⁴⁾. One specfic target appears to be the self protein BP180, which is a key anchoring protein in the skin and the nasal mucosa. Anti-BP180 antibodies were elevated in CRSwNP patients, suggesting autoimmune targeting of the epithelial barrier⁽³⁴³⁾.

Local IgE has been the most commonly implicated antibody in the pathophysiology of CRSwNP. Elevated IgE levels in nasal polyps have been shown to be independent of systemic atopy, driven by BAFF with local class switching, predict poor progno-sis and correlate with the presence of IgE against staphylococcal superantigenic toxins. These toxins have the capacity to act as both B and T cell superantigens in nasal polyps, fostering a poly-clonal IgE response⁽³⁹⁰⁾. Studies have demonstrated that the IgE in nasal polyps is polyclonal and can trigger mast cell

degranula-tion, suggesting a significant role for IgE in the pathophysiology of this subset of CRSwNP patients⁽³⁹¹⁾. The therapeutic potential of anti-IgE for nasal polyposis has recently been demonstrated in CRSwNP patients as well^{(313, 384)}.

Similar to IgE, IgG4 is a minor IgG subtype whose production is also regulated by Type 2 (IL-4 and IL-13) cytokines. Local IgG4 expression is high in serve eosinophilic CRS, with higher ILC2, plasma cells, B cells and activated CD8+ T cells^{(362, 363)}. The complement system is a collection of circulating and cell membrane proteins that play important roles in host defence against microbes and in antibody-mediated tissue injury⁽²⁷⁴⁾. Complement assists the antibody-mediated lysis of microbes but can result in tissue injury, particularly in cases of autoimmu-nity. At this point, there is only weak evidence to support a com-plement defect associated with CRS pathophysiology^(366-368). The evidence for excessive complement activation in CRS is stronger, suggesting the importance of antibody-mediated events in CRS pathophysiology possibly related to autoimmunity⁽³⁸⁴⁾.