Dendritic cells as critical players in allergic rhinitis

Abstract

More than three decades ago, the presence of dendritic cells (DCs) was described in the nasal mucosa. From then on, there was a gradual increase of knowledge on the basic aspects of DC function. DCs in the airways sample the airway lumen for incoming antigen and present it to the various players of the immune system subsequently, so that an optimal response is induced to clear pathogens. The enormous interest of immunologists in the role that DCs play in the induction and regulation of immune responses is currently beginning to integrate into the framework of disease pathogenesis. DCs have been implicated in causing allergic sensitization as well as ongoing allergic inflammation. DCs play a crucial role in the development and maintenance of allergic rhinitis (AR). The depletion of DCs in AR abrogates the features of AR. Consequently, DCs and several molecules on DCs are ideal targets for therapeutic intervention.

• allergic rhinitis
• epithelium
• myeloid dendritic cell
• plasmacytoid dendritic cell
• thymic stromal lymphopoietin

The nose is the part of the human body with mucosal tissue that gets in contact with inhaled air. It functions like an air conditioner that filters air that is inhaled. Because of this function, the nose is continuously exposed to pathogens. These dangers need to be prevented by good defense mechanisms that protect against pathogens. Epithelial cells form the first cellular barrier, with an important barrier role for tight junctions in between epithelial cells. In cases in which this is insufficient, there is another defense mechanism caused by immune cells. These immune cells stay in the epithelium until they have been activated by a stimulus that is recognized by that particular cell. The most important immune-competent cells in the epithelium are the dendritic cells (DCs). DCs play an important role in allergic inflammation that was first described by Fokkens et al. (1, 2). In steady-state condition, a few of these professional antigen presenting cells (APCs) are present (1, 2). These DCs form an intensive network of cells in between epithelial cells 3–(5). Intraepithelial location of DCs is often seen. The DCs seem to be anchored in the epithelium by the use of tight junction protein claudin-1, and DCs can penetrate beyond the most apical tight junction protein, occludin. DCs are able to open and make connections with the tight junctions, enabling dendrite sprouting beyond the epithelium (6). The tight junction mechanisms behind have two important advantages: 1) the DC itself is anchored; and, due to the penetration, 2) the epithelial tight junction barrier stays the same (7, 8).

Triggers of DC activation in the nasal mucosa

When a DC is triggered, it gets activated and changes from an immature DC into a mature DC phenotype. The sentinel function of DCs is very important for inducing an immune response. The (in)direct activation of DCs occurs due to the so-called pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (9). Both epithelial cells and DCs express a broad range of PAMPs and DAMPs [for a review, see Willart et al. (10)] for the epithelium, with the consequence of cytokine production and release that can activate the innate immune system, including DCs. Important chemokines are the so-called alarmins (11), including cytokines like IL-33, thymic stromal lymphopoietin (TSLP), and other members of the interleukin (IL)-1 family, including IL-1α, IL-1β, and the IL-7 family (Fig. 1) (12). TSLP, IL-33, and IL-33R–T1ST2 have been associated with asthma genome-wide, but typically the impact of each of these genes is mild 13–(15). The effect of these alarmins in general is the activation of DCs, with the consequence of migration to the draining lymph node (LN). IL-33 is one of the epithelial-derived cytokines that supports Th2 immunity via the binding of the ST2 receptor that is detectable on immune-competent cells, including DCs, in the nasal mucosa (16, 17). This receptor is also expressed by DCs, and activation of IL-33 leads to increased expression of MHCII, co-stimulatory molecules, and proinflammatory cytokines, chemokines (including CCL17, CCL22) and support the Th2 response 18–(20). IL-33 is detectable in the nasal mucosa of allergic rhinitis (AR) patients and elevated in allergen-challenged AR patients (21). Moreover, IL-33 plays a critical role in experimental AR (22). IL-25 (IL17E), which has similar functions as IL-33, is important in the intestine (23) and is not crucial for the development of AR (12). Also, TSLP is expressed in the nasal mucosa of AR and chronic rhinosinusitis patients (24), and it is important to stimulate DCs in AR (25). Moreover, TSLP-activated DCs play a role to induce naïve CD4+ T-cell differentiation into Th2 cells (26).

Fig. 1. Dendritic cells as critical players and sentinels of the immune system in the nasal mucosa of allergic rhinitis. The presence of DCs in the epithelium fulfills a sentinel function in the steady-state condition. During inflammation and allergic inflammation, DCs were activated due to the so-called alarmins: TSLP and members of the IL-1 family, including IL-1α and IL-33. Also, nonphysiological factors like staphylococcal enterotoxin B antigens act as super-antigens, influencing the epithelium and indirect DCs. Furthermore, the nonphysiological presence of allergens like house dust mites (HDMs) and the cockroach allergen Bla G 7 influences the epithelium and DCs. Once epithelial DCs are triggered, they get activated and migrate from the epithelium via the lymph vessel to the draining lymph node. Moreover, activated DCs are also present in the lamina propria and are seen in close proximity to T-cells, suggesting the support of a local immune response. An important molecule for DC migration is CCR7.

The presence of certain antigens could influence the number of DCs. The superantigen staphylococcal enterotoxin (SEB) in the nasal mucosa of AR impairs the numbers of tolerogenic DCs. In this active process, the epithelial integrin Avb6 plays a role. Experimental confirmation of this relation is seen in experiments in which neutralization of SEB by antibodies in epithelial culture experiments could impair the suppressive effect of SEB on Avb6 levels of mRNA and protein (27).

There are also examples of direct DC activation by allergens. In the case of cockroach allergy in AR patients, it became clear that the cockroach allergen rBla g 7 induces DC-dictated Th2 polarization of CD4+ T cells through a mechanism in which TIM4 is involved (28). Moreover, rBla g 7 induces IL-13 production of human DCs, which has implications for the isotype switch of B-cells into immunoglobulin E (IgE)-producing B-cells (28).

DC migration from peripheral tissue to the draining LN

Once DCs have been activated, they express receptors reacting to chemokine signaling. Then, DCs start to migrate in the direction of an LN. Data strongly suggest that these activated maturated DCs migrate via the lymphatic vessel and appear in the draining LN. From textbooks, it is known that when DCs are activated, they express CCR7, which is important for the migration from peripheral tissue sites toward the draining LN. Blocking CCR7 impairs the pathogenic contribution of DCs in allergy (29). Once they have arrived in the draining LN, they present antigen to antigen-specific T-cells and decide in which of them the immune response should occur. This process is generally the same for all immune responses.

Studying the deposition of airborne particles indicated that more than 80% are trapped in the nasal mucosa, and the minority is trapped in the lower airway (30). Dyes used as models to study flow in the nasal mucosa illustrated that dyes could not penetrate the nasal mucosa easily, revealing the physical barrier function of the nasal mucosa 31–(34). Introduction of carboxyfluorescein succinimidyl ester (CFSE)-labeled DCs or DiY Dye in the nasal mucosa in droplet form resulted in detectable CFSE-labeled DCs and DiY in the nasal mucosa in the nasal-associated lymphoid tissue (NALT) and in the cervical LN, which drains the nose. No CFSE-labeled cells could be detected in either the mediastinal LN or the lung (35). In a human migration study of the respiratory tract, it is indicated that antigen pulsed DCs that are introduced by injection in the nasal inferior turbinates can be traced back to the regional LNs (36). Although DCs used in this study were monocyte-derived DCs, they still clearly show a localized draining and migration pattern of the introduced DCs, as observed with the DiY and allergen-loaded DCs in murine studies (4).

Nasal mucosa dendritic cell type and quantification

The use of markers for DCs includes CD1a as one of the most important characteristic markers. Other markers for myeloid DCs (mDCs) are CD1c, MHCII, CD11c, CD207, and CD208, and markers for plasmacytoid DCs (pDCs) are CD123, CD303, and CD304. Several of these markers were widely in use as blood DC markers. For histology, the use of an appropriate marker along with a dendritic morphology is essential to characterize cells as DCs (4) (Table 1). This is important because the markers used are often expressed on other cell types as well. Ratio calculations between mDCs and pDCs were performed to reflect active allergic inflammation and/or a steady state in nasal mucosa tissue (37). Moreover, an active role for pDCs in allergic inflammation was also confirmed in allergen challenge studies with an important role for the DC stimulation of TNFα (38). Elevated numbers of DCs (CD209 DC-Sign) were observed in allergic fungal rhinosinusitis (39). Models of AR showed an elevated number of DCs (CD11c-positive cells with a dendritic morphology) (4, 40, 41). Moreover, the tolerogenic number of DCs as measured as CD11c+ ADLH1/2+ cells was shown to be reduced in AR mice, and the corresponding number of FoxP3 shows the same reduction, indicating an impaired tolerogenic response in AR mice (41). A nice example of increased levels of HLADR+ CD209+ CD14+ DCs of other nasal disease pathogens is seen in the circulation of rhinitis patients with or without nasal polyps. Moreover, the proportion of CD4 +CCR8+ T-cells reflecting Th2 cells is higher in rhinitis when compared to controls (42). Moreover, this observation could be confirmed in the nasal mucosa tissue of allergic fungal rhinosinusitis patients (39).

Table 1 Human DC antibodies

Markers	Clone (preferable clone)	mDCs	pDCs	Other cells	Suitable for histology
CD1a	okt6	+++	−	Immature T-cells (thymus)	+++
CD11c	SHCL-3	+++	+	Monocytes, macrophages, and neutrophils	+++
HLADR		+++	+	B-cells, activated T-cells, monocytes, mast cells, and epithelial cells	+++
CD207	DCGM4	++	−	−	++
CD208	104.G4	++	−	−	++
CD209		+	+	Macrophages	++
CD1c BDCA1		+++	−	Blood DCs	+
CD303 BDCA2		−	+++		±
CD141 BDCA3		+++	−		±
CD304 BDCA4		−	+++		−
CD14		±	±	Monocytes	++
CD123	9F5	−	+++	Vessels	++

DC, dendritic cell; mDCs, myeloid DCs; pDCs, plasmacytoid DCs.

Nasal mucosal mDCs and pDCs numbers were significant increase in relevant allergen challenged nasal mucosa in AR which was not seen in antigen-challenged control subjects at 8 hours following allergen challenge. In the skin of AR patients only an increase in mDCs was observed 48 h post allergen challenge and not for pDCs. Allergen challenge induces the production of IL-10 in nasal mucosa of control subject which was not seen in AR (43). Tolerogenic DCs identified as CD11c+ transforming growth factor (TGF)-β-positive cells were less present in the nasal mucosa of AR patients when compared to non-AR controls. Along this, the Foxp3+ CD4+CD3+ T-cells were also less present in the nasal mucosa of AR patients when compared to non-AR controls. The levels of IL4 and IgE in nasal mucosa tissue homogenates were higher in AR patients than in non-allergics. The number of mast cells was higher in AR patients than in non-allergics (26). The aforementioned examples illustrate the close correlation between disease pathogenesis DCs and Th2 cells.

DCs express immunologically relevant molecules; therapeutic targets

Myeloid DCs from AR were constitutively primed to induce Th2 cytokines and TNF in allogenic CD4+ T cells, whereas no differences were observed for interferon (IFN)-γ or IL-10 (44). These DCs, obtained from either human blood or nasal mucosa tissue DCs, express lower levels of the ICOS ligand in AR than in controls. ICOS is a co-stimulatory molecule on T-cells that is important for a stable DC–T-cell interaction; it is associated with Th1 response, and the expression of ICOS on DCs is reduced in allergic inflammation models (45). Regarding the cytokine production by DCs, no differences in DC-derived IL-10 and IL-12 were seen between AR and control mDCs. Targeting by blocking antibodies of ICOSL in control DCs upregulated IL-13 but not IFN-γ in co-cultures with T-cells, whereas PD-L1 blockade upregulated both IL-13 and IFN-γ (44). OX40 ligand (OX40l) has already been long known as a co-stimulatory molecule. OX40l plays a sentinel role in the adaptive immune response by promoting Th2 polarization of naïve T cells within the LN. The OX40–OX40l axis might be an interesting treatment opportunity; although the first in vivo trails were not as good as expected for all patients, in some patients it seemed to be beneficial (46, 47). Some recent studies targeting indirect DCs with antibodies against OX40l or TSLP in asthma might have also beneficial effects on AR (47, 48) (Table 2).

Table 2 Novel DC targets in allergic rhinitis

Compound or molecule	Effects on DCs	Inflammation
FTY720	Impaired DC migration capacity and impaired T-cell function	Reduced allergic airway inflammation
OX40L	Impaired DC migration	Reduced allergic airway inflammation
TSLP	Prevents TSLP binding on the TSLPr of DCs and impaired activation of DCs	Reduced allergic inflammation
Notch pathway members	Prevent interaction between DCs and T-cells and impaired T-cell function	Reduced allergic inflammation

DC, dendritic cell; TSLP, thymic stromal lymphopoietin; TSLPr, TSLP receptor.

Pharmacological interaction of in vitro cultured DCs in the presence of the Clara cell 10-kDa (CC10) showed reduced levels of OX40l expression, lowered the production of IL-23 and IL-6, and enhanced the expression of CD86 and the production of TGF-β. DCs treated with CC10 protein inhibit Th17 responses through modulating DCs in the setting of AR (49).

Sphingosine 1 phosphate (S1P) and its analogs are important mediators for immune cell migration. DCs express S1P receptors 1–5 (S1P1–5). Sphingosine lyase (SL) is important for the breakdown of S1P; FTY720 is an inhibitor of SL and an analog of S1P. S1P reduces the migration and the capacity of the antigen presentation function of DCs (50). Topical intranasal treatment of FTY720 may be mediated by affecting and impairing the DC function. We found that topical treatment with FTY720 reduces the features of AR. When OVA antigen was applied intranasally, OVA-specific T-cell divisions were seen in the lymphoid structures draining the upper airway, including the NALT. The division was blocked partially in the NALT by FTY720 treatment when compared to saline treatment. This effect was not observed in the cervical nodes. This illustrates the functional effect of FTY720 on DC migration and also might influence the type of Th immune response (40). Moreover, T-cell differentiation pursuant to treatment with S1P (40) is such that Th17 cell development is almost unaffected, whereas the outgrowth of other Th populations is reduced (51). In our hands, FTY720 showed a significant reduction in IL-4-producing CD4-positive T-cells cultured under Th2 conditions when FTY720 was added to the culture. IFN-γ-producing CD4 T-cells and IL-17-producing Th17 cells were not affected by FTY720 treatment (40).

Recent novelties; Lessens from outside the nasal mucosa

In the development of allergen-specific immunotherapy, several advantages were performed in preclinical and clinical research. Due to the use of adjuvants like toll-like receptor agonist and vectors like liposomes and microspheres in clinical trials, advantages were seen in Phase II and Phase III trials (52). Nasal mucosa administration of antigens in the presence of adjuvants like nano-emulsion induced distinct pathways of humoral and cellular immunity (53).

CD40, CD80, and CD86 are molecules expressed by DCs that are important for a well-established immune response. Recently accumulating data appeared from the Notch pathway. Notch ligands expressed by DCs are important players for the outcome of immune responses. The DC Notch ligand molecules Jagged 1 and Jagged 2 were important for Th2 responses, whereas delta-like ligand 1 and 4 were important for Th1 responses (54). In vivo data by our group in asthma models indicate the importance of the Notch pathway. Mice that lack recombination signal binding protein for the immunoglobulin kappa J region (RBPj), which is an important protein of the intracellular Notch complex that binds Gata3, showed impaired allergic airway inflammation, including less eosinophilic inflammation, lower levels of Th2 cytokines in BAL fluid, and lower levels of IgE. This implies that the Notch pathway is important in allergic airway inflammation.

Conclusions

DCs together with epithelial cells play an important role in the connection between innate and adaptive immune responses of the upper airways. The DCs are the migratory cells that ideally could be targeted by inhibiting their migratory capacity and by therapeutically preventing their interaction with cells of the adaptive immune system.

Conflict of interest and funding

The author declares no conflict of interest and funding.