The SP‐TLR axis, which locally primes the nasal mucosa, is impeded in patients with allergic rhinitis

Abstract Background Substance P (SP) and toll‐like receptors (TLRs) contribute to airway disease, particularly during viral infection. We recently demonstrated that SP can act as an initial response to viral stimuli in the upper airway by upregulating TLRs in the nasal epithelia (the SP‐TLR axis). Patients with allergic rhinitis (AR) suffer from prolonged airway infections. The aim of the present study was to examine if patients with AR exhibit a disturbance in the SP‐TLR axis. Method Human nasal biopsies and human nasal epithelial cells (HNEC) from healthy volunteers and patients with AR were cultured in the presence of SP. Epithelial expression of TLR4, neutral endopeptidase (NEP) and neurokinin 1 (NK1) were evaluated with flow cytometry and/or quantitative polymerase chain reaction after 30 min to 24 h. The effect of SP on nasal lipopolysaccharide‐induced interleukin‐8 (IL‐8) release was investigated. Results SP stimulation of tissue from healthy volunteers resulted in a transient increase of the TLR4 expression, whereas stimulation of AR patient‐derived material led to a delayed and prolonged upregulation of TLR4. NEP expression in HNEC was lower in AR than healthy controls whereas NK1 receptor expression was increased. SP pretreatment increased TLR4‐dependent IL‐8 expression in healthy controls, but not in AR. Conclusions SP‐induced regulation of TLR4 in the human nasal mucosa is disturbed in AR. An altered SP‐mediated innate immune response may contribute to the dysfunctional and often prolonged responses to infection in AR.

alterations in the innate immune response in atopic airway diseases, leading to susceptibility to infections. 2,3 In particular, expression levels and function of toll-like receptors (TLRs), as early responders to viral and bacterial particles, have been shown to be altered. [4][5][6][7] Substance P (SP) is known to be locally induced upon nasal allergen challenge 8 and its upregulation in symptomatic AR is welldocumented. 9 As a mediator of neurogenic inflammation, SP induces vasodilation, vascular leakage, mucous secretion, and inflammatory cell recruitment, all contributing to the hallmark symptoms of AR. 10 In addition to its role in neurogenic inflammation, recent studies have highlighted a function for SP as mediator of innate immunity. SP has been shown to contribute to the development of an antiviral innate immune response in mice, 11 as well as regulate messenger RNA (mRNA) levels of TLRs in human mast cells. 12 We recently published a study outlining a novel role for SP as a regulator of the innate immune system in the nasal mucosa. SP, released following stimulation with the viral mimetic and TLR7 agonist R837 induced a rapid increase in the expression of several epithelial TLRs including TLR3 and TLR4. 13 Hence, it is tempting to conclude that this SP-TLR axis primes the nasal mucosa for incoming infections.
The action of SP is known to be controlled by a number of mechanisms. First, SP is broken down by a variety of peptidases, most notably neutral endopeptidase (NEP). 14 NEP exists in a soluble and membrane-bound form and is expressed on epithelial cells, vessels and serous cells of submucosal glands of the nasal mucosa. 15 NEP is released within 20 min after a nasal allergen challenge, 15 and it is known to limit the concentration of SP that reaches its receptor as inhibition of NEP potentiates the action of SP in the human nasal mucosa. 16 Second, that action of SP is controlled by its cognate receptor neurokinin 1 (NK1). Increased expression of NK-1, as seen in asthmatic airways, 17 increases the effect of SP. However, upon SP--NK-1 interaction, the agonist-receptor complex is internalized, limiting further and continued action of SP. 18 As AR is associated with alterations in the expression of SP, the present study was designed to compare elements in SP-TLR axis in nasal biopsies and epithelial cells derived from patients with AR with corresponding material from healthy individuals.

| Ethics statement
The study was approved by the local Ethical Committee, Stockholm, Sweden (number 2014/299 and 2016/826). All participants gave their written, informed consent. All procedures were conducted according to the principles expressed in the Declaration of Helsinki.

| Subjects and study design
The study included 30 nonsmoking, non-astmatic, patients with moderate to severe birch and/or grass pollen-induced seasonal AR, and 35 healthy volunteers. Human nasal epithelial cells (HNEC) were isolated from 24 healthy subjects and 22 AR patients; nasal biopsies were isolated from 11 healthy subjects and 8 AR patients. All samples were acquired outside the pollen season. Patient characteristics are listed in Table 1.
The diagnosis of birch or grass pollen-induced AR was based on a positive history of seasonal AR as well as a positive ImmunoCap Rapid (Asthma/Rhinitis Adult) test (Phadia; Thermo Fisher Scientific).
Patients who did not meet both criteria were excluded from the study. The ImmunoCap Rapid (Asthma/Rhinitis Adult) assesses the presence of circulating specific immunoglobulin E against 10 common airborne allergens, including pollen (birch, timothy, mugwort, olive, wall pellitory), house dust mites (HDM), mould and common animal allergens (cat, dog, cockroach). Exclusion criteria included a history of upper airway infection within 2 weeks prior to visit, treatment with local or systemic corticosteroids within 2 months before the visit, and history of chronic rhinosinusitis with or without presence of nasal polyposis.
Five AR patients tested positive for allergy towards common animals allergens and one AR patient tested positive for allergy towards HDM. Two AR patients had comorbid allergic asthma.

| Isolation and culture of human nasal biopsies
Human nasal biopsies (HNBs) were isolated as described previously. 19 Two biopsies per patient were obtained from the inferior

| Multiplex quantitative PCR
Real-time multiplex qPCR was performed using a dual-labelled probe technique with the Quantifast Multiplex PCR + R kit (Qiagen) on a Stratagene Mx3000P Cycler (Agilent Technologies). All reactions were run with the following program: activation at 95°C for 5 min, followed by 40 cycles of two-step cycling at 95°C for 45 s and 60°C for 45 s. Primers and probes for reference genes (actb, gapdh) and target genes (tlr4) were acquired from Sigma-Aldrich and are detailed in Table 2. Taqman primers for cxcl8 (Hs00174103_m1) were acquired from Applied Biosystems. Analysis of C t values was performed using the ΔΔC t method.

| Statistical analysis
Data were analysed with GraphPad Prism software (Version 6.0).
Results are expressed as mean ± SEM. In all experiments, n is equal to the number of subjects. Analysis was performed using a two-way analysis of variance, followed by a Fisher's posttest. A p value of 0.05 or less was considered statistically significant.

| R837 induces SP release in the nasal mucosa
To ascertain if the previously demonstrated R837-induced SP release in nasal mucosa was affected by the allergic condition per se, HNBs and HNECs from healthy volunteers and patients with AR were stimulated with R837. SP release was unaffected by the allergic versus non-allergic status, neither in the HNB specimens (p = 0.9), nor in the HNEC material (p = 0.07; Figure S1). was seen in AR samples, but with a later TLR4 peak ( Figure 1A,B), demonstrating significant increases at 240 min in HNB (p = 0.047).

| The SP-induced TLR4 upregulation in the nasal mucosa is delayed in AR
TLR4 increases were significantly higher in HNB from AR patients at 240 min (p = 0.029) and in HNEC at 60 min (p = 0.046), as compared to respective material from healthy volunteers. Also, at the mRNA level, after 5-24 h, the TLR4 upregulation appeared earlier and was higher in HNECs from healthy individuals than in material from patients with AR ( Figure 1C); at 4 hours tlr4 mRNA was significantly upregulated in healthy controls (p = 0.016), and significantly elevated above values in AR patients (p = 0.032).

| Decreased NEP expression in AR
NEP expression was found in HNEC and HNB (Figures S2 and S3).
NEP levels were analysed in HNBs and HNEC during basal conditions as well as after stimulation with SP. No differences in baseline expression of NEP were evident in whole biopsies (Figure 2A, However, when only epithelial cells of the same biopsies were analysed, the NEP levels appeared to be lower in specimens from allergic patients ( Figure 2B, p = 0.618). This difference was more marked, reaching significance (p = 0.0003) when the NEP analysis was repeated on the HNEC ( Figure 2C). SP stimulation of the HNBs resulted in a decline in NEP expression in nasal biopsies. This reduction appeared to be more pronounced in biopsies from allergic individuals, reaching significance at 240 min (p = 0.04) (Figure 2A). SP stimulation had no effect on NEP expression in epithelial cells from nasal biopsies from healthy controls, whereas a marked increase in NEP was evident 240 min after stimulation in allergic individuals (p = 0.009; Figure 2B). The levels of NEP in HNEC seemed to be unaffected by SP, with the differences in levels observed during the basal conditions being maintained ( Figure 2C).

| Increased NK-1 receptor expression in AR
The effect of SP on TLR4 up-regulation is mediated via NK-1 receptors (NK1R) on the surface of epithelial cells. 13 To assess whether the NK1R was affected in AR, its expression level was analysed with flow cytometry in HNBs and HNEC after stimulation with SP.

| Pretreatment with SP does not prime lipopolysaccharide mediated IL-8 release in AR
TLR4 is responsible for inducing early inflammatory responses against bacterial lipoproteins, such as lipopolysaccharide (LPS). In order to determine whether altered expression of TLR4 following SP stimulation resulted in altered responses to bacterial exposure, isolated HNEC were stimulated with SP for 30 or 60 min and then subsequently exposed to LPS. When the levels of IL-8 mRNA were assessed 3 h after the LPS exposure, increases were evident in the control, nonallergic material, but failed to appear in cells from patients with AR ( Figure 4).

| DISCUSSION
The present study demonstrates that the ability for SP to regulate innate immunity is impaired in patients with AR, with a heightened and prolonged upregulation of TLR4, and an associated lack of The present study demonstrates that SP rapidly, and transiently, upregulates nasal epithelial TLR4 in isolated cultures, as well as in a multicellular system, represented by nasal biopsies. This corroborates our previous findings 13  NEP is known to break down SP and limit its actions. 14 As previously described, 15  Despite the delayed and prolonged increase in TLR4 in AR individuals, changes to LPS-induced proinflammatory response were absent. Whereas in healthy individuals, SP upregulated transcription of cxcl8 following LPS stimulation, no priming effect was evident in AR. The reason for this difference in function is unknown. One study has highlighted that upregulated TLR4 in allergy is associated with increased levels of Th2 and anti-inflammatory cytokines. 5 Therefore, sustained upregulation of TLR4 in allergy may lead to pronged release of pro-allergy factors (e.g., IL-13 or IL-4), rather than typical proinflammatory cytokines.
Alterations in TLR4 and SP expression are well-documented in AR. Levels of SP are consistently upregulated, 26 which may in part be related to a decrease in epithelial NEP expression highlighted in this study. NK1 has also been shown to be upregulated in atopic airways. 17 Most notably, TLR4 expression is upregulated in symptomatic and persistent AR, 5,27  The present study demonstrates that in healthy individuals, SP functions as a regulator of innate immunity, particularly in upregulation TLR4 and thus priming LPS-induced inflammation. In patients with AR this SP-TLR axis appears to be impaired, with a heightened and prolonged upregulation of TLR4, associated with the lack of the expected increased response to LPS. This compromised ability for SP to regulate immunity may, at least in part, contribute to the more severe and prolonged infection-induced diseases often seen in conjunction with airway allergy.
LARSSON ET AL.