Increased nasal matrix metalloproteinase-1 and -9 expression in smokers with chronic rhinosinusitis and asthma

A potential mechanism underlying cigarette smoke-induced airway disease is insufficient tissue repair via altered production of matrix metalloproteinases (MMPs). Osteitis is a signature feature of recalcitrant chronic rhinosinusitis (CRS) and often results in revision surgery. The present study aimed to investigate MMP expression in the nasal tissues of asthmatic patients with CRS and any association with cigarette smoking and osteitis. Thirteen smokers with CRS and asthma, 16 non-smokers with CRS and asthma, and seven non-smoker asthmatic patients without CRS were prospectively recruited. The expression of MMPs and associated immunological factors in surgically-obtained nasal tissues was evaluated via real-time PCR and western blotting. Maximal bone thickness of the anterior ethmoid (AE) partition was measured in axial sinus computed tomography (CT) sections. MMP-1 and MMP-9 expression was increased in the nasal tissues of smokers with asthma and CRS via real-time PCR and western blot. Maximal AE partition bone thickness was greater in smokers with CRS and asthma than in non-smokers with CRS and asthma. MMP-1 and MMP-9 levels were correlated with maximal AE bone thickness. Cigarette smoking was associated with the up-regulation of MMP-1 and MMP-9 in the nasal tissues of patients with airway inflammatory diseases, and with AE osteitis, and with therapeutic resistence.

Chronic rhinosinusitis (CRS) and asthma are common inflammatory airway diseases and frequently comorbid, as per the "unified airway" concept 1 . Type 2 inflammatory cytokines, such as IL-5 and IL-13, are considered key drivers of airway inflammation in patients with CRS and asthma 2,3 . However, the IL-17A mediated immune response has been linked to neutrophil recruitment, airway remodeling, and resistance to corticosteroid-based therapy in airway disease cases [4][5][6] . A recent study by our group revealed that cigarette smoking was related to IL-17A activation in the nasal tissues of asthmatic patients with CRS and attenuated improvements in asthmatic patients after nasal surgery 7,8 . Cigarette smoke contains over a 1,000 chemicals, and chronic obstructive pulmonary disease (COPD)-like airway injury and remodeling occur in chronic cigarette smoke exposure rodents models 9,10 . A potential mechanism for cigarette smoke-induced airway disease is insufficient tissue repair via altered production of matrix metalloproteinases (MMPs) 11 .
MMPs comprise a family of Ca 2+ -activated, zinc-dependent endopeptidases, which may be produced by airway epithelial cells, fibroblasts, and inflammatory cells 11,12 . MMPs play an essential role in the degradation of basement membranes and extracellular matrix proteins including collagens IV, V, VII, X, and XIV; gelatin; and elastin. MMPs contribute to the tissue edema and remodeling seen frequently in inflammatory diseases of the airway including asthma, COPD, and CRS 11,13,14 .
Tissue remodeling in the lower airway has been extensively studied in asthmatics and in patients with COPD. For example, MMP-9, also known as gelatinase B or 92 kDa gelatinase, is a critical elastolytic enzyme produced by alveolar macrophages in COPD patients. It is also secreted by neutrophils, epithelial cells, mast cells, and fibroblasts 15 . Higher levels of MMP-9 are associated with increased asthma severity and decreased lung functioning. MMP-12 is also an elastolytic proteinase found in the alveolar macrophages of cigarette smokers 16 and is relevant to cigarette smoke-induced emphysema 17 .
Recent studies have indicated that sinonasal tissue remodeling occurs secondary to CRS 18 . Elevated MMP-1,-2,-7,-8, and -9 have also been noted in the sinonasal tissues of CRS patients 14,19,20 . In particular, MMP-9 is significantly lower in patients with good mucosal healing after sinus surgery compared to those with poor healing 21 . MMP-9 plays an important role in the pathophysiology of osteitis in CRS 22 . Osteitis, a form of neo-osteogenesis due to inflammation rather than infection, is a common factor in recalcitrant CRS and is associated with revision sinus surgeries and CRS severity, indicated by elevated Lund-Mackay computed tomography (CT) scores [23][24][25] . IL-17A, a signature CRS cytokine 26,27 , promotes MMP-9 expression by activating the NF-κB signaling pathway in the nasal tissues of patients with CRS and nasal polyps 28 . As a result, the relationship between cigarette smoking, IL-17A, and MMPs is worthy of investigation.
Given the above background, we hypothesized that IL-17A-mediated immune responses and MMPs expression might be associated with cigarette smoke-related airway inflammation, osteitis formation, and resistance to current therapeutic regimens. The present study aimed to investigate the expression of MMPs in the nasal tissues of asthmatic patients with CRS and its association with cigarette smoking and osteitis.  29 and CRS was defined using the criteria established in the European CRS position paper 30 and was based on subjective symptoms and objective findings from nasal endoscopy and sinus CT. Participant inclusion criteria included a diagnosis of asthma (1) with regular follow-up for at least 1 year; (2) failed medical treatment for nasal symptoms; and (3) plans to undergo nasal surgery. Exclusion criteria included previous nasal surgeries or major medical comorbidities such as diabetes, nephrotic diseases, autoimmune disorders, immunodeficiencies, malignancies, and other chronic illnesses. Seven non-smoker asthmatic patients without CRS (confirmed by sinus CT) were enrolled in the control group during septomeatoplasty and/or turbinoplasty. Participants provided informed consent prior to study enrollment. All research was performed in accordance with the relevant guidelines and regulations and all Institutional Review Board requirements.
Patient clinical characteristics were determined at enrollment and at a 6 month follow-up appointment. Patients were asked if they regularly used cigarettes or other forms of tobacco pre-operatively. Patients were considered to be smokers if they reported regular or on-going smoking in the last 12 months and non-smokers were considered to be those who never regularly used cigarettes 31 .
A preoperative nasal endoscopy examination and sinus CT, which were scored using the Lund-Kennedy endoscopy score and Lund-Mackay CT score, were completed in all patients. The maximal thickness of the bony partition of the anterior ethmoid sinuses (AE) was measured on an axial sinus CT section for each patient (Fig. 1). Pulmonary function tests, including forced vital capacity (FVC), forced expiratory volume in 1 second (FEV 1 ), and FEV 1 /FVC ratio, were measured before and 6 months after nasal surgery. The severity of sinonasal symptoms and the level of asthma control were evaluated by the Sino-Nasal Outcome Test-22 (SNOT-22) 32 and Asthma Control Test (ACT) questionnaires 33 , respectively. collection and processing of specimens. Nasal mucosal specimens were obtained during nasal surgery and rinsed in phosphate-buffered saline (pH 7.6), stored in liquid nitrogen at −70 °C, and then processed for immunoblotting and real-time polymerase chain reaction (PCR).
Total RNA was extracted from nasal specimens using the RNeasy Mini Kit (Qiagen, Strasse, Germany) and quantified by NanoDrop (Thermo Scientific, Barrington, Ill). Reverse transcription was performed with random hexamer primers using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was done using the TaqMan assay with primers specific to each target gene (Table 1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal housekeeping control gene. The Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems) was used. The amplification conditions were an initial incubation at 95 °C for 10 min; 45 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 10 s; and final cooling to 40 °C. Each sample was run in triplicate. All triplicate mean threshold cycle (Ct) values were normalized to GAPDH to determine the ΔCt values and relative mRNA levels for each target gene were calculated using the ΔΔCt method.
Western blot analysis of MMps. Nasal tissues from 21 participants with adequate specimens, including seven smokers with asthma and CRS, nine non-smokers with asthma and CRS, and five asthmatic non-smokers without CRS were processed for western blot analyses of MMPs. Total protein was prepared using a 1% IGEPAL lysis buffer  Table 2. There were no differences in participant age, severity of sinusitis, presence of nasal polyps, and lung function between smokers and non-smokers with asthma and CRS. Sex differed between the groups, with significantly more males in the smoker group (P < 0.01).
Protein analyzed by western blotting confirmed the results of mRNA and revealed that smokers with CRS and asthma had higher levels of MMP-1 (Fig. 3a,b, P = 0.032) and MMP-9 (Fig. 3a,c, P = 0.042) expression than those of non-smokers with CRS and asthma. There was no difference in MMP-12 expression between the groups (Fig. 3d, P = 0.418).

Discussion
The present is the first study to evaluate the link between cigarette smoke and AE neo-osteogenesis in patients with asthma and CRS in terms of IL-17A and MMP expression. Our results revealed the expression of MMP-1, MMP-9 in the nasal tissues, and maximal AE bone thickness on CT scans were greater in smokers with asthma and CRS than in asthmatic non-smokers with CRS and asthmatic non-smokers without CRS. Tissue MMP-9 mRNA levels were correlated with IL-17A and the transcription factor AhR. In addition, MMP-1 and MMP-9 protein levels were correlated with maximal AE bone thickness on CT scans.
Osteitis was initially defined as bony thickening of the sinus walls in CRS 34 . The histopathology of osteitis in primary CRS is thought to involve neo-osteogenesis and bone remodeling, which occurs secondary to tissue inflammation 25 . Recent studies have found that osteitis is a characteristic of recalcitrant CRS and is associated with increased disease severity and the need for multiple surgeries 23,24 . Previous studies have also revealed that exposure to smoking diminishes long-term outcomes and increases the risk for revision surgery following endoscopic sinus surgery 31,35 . In the present study, we found a link (involving IL-17A, MMP-1, and MMP-9 expression) between cigarette smoke and neo-osteogenesis in AE with asthma and CRS. Taken together, these results reveal that cigarette smoking may have association with an IL-17A-mediated inflammatory response and increased MMP-1 and MMP-9 expression in the nasal mucosa of patients with asthma and CRS. This may then results in tissue remodeling, which involves neo-osteogenesis or osteitis of the sinus, and thus poorer outcomes following sinus surgery.
Cigarette smoke contains many AhR-activating ligands such as polycyclic aromatic hydrocarbons (PAHs) 36,37 . Exposure to diesel exhaust particles, traffic-related air pollution, and PAH induces IL-17A production and can lead to severe asthma 38 . Th17 polarization is initiated in an AhR-dependent manner through the stimulation of atmospheric particulate matter (PM) in the murine lung. PAHs in the PM are likely sources of Th17-promoting activity 39 . Recent work also demonstrated that IL-17A promotes the expression of MMP-9 by activating the NF-κB signal pathway, revealing a crucial role for IL-17A in the pathogenesis of CRS and associated tissue remodeling 28 . MMP-9 also plays a critical role in the pathophysiology of osteitis in CRS and MMP-9 overexpression, which is steroid-independent, suggesting its relevance to therapeutic resistance 22 . Taken together, MMPs from IL-17A-mediated inflammation via the AhR activation induced by cigarette smoke exposure may thus contribute to neo-osteogenesis and recalcitrant disease in patients with CRS and asthma.
In the current study, MMP-1 and MMP-9 levels were correlated with FEV1/FVC (% predicted), while IL-5 and IL-13 mRNA levels were associated with the CT scores and endoscopy scores, respectively. These results implicate MMPs, which might be induced by cigarette smoke, in lower airway obstruction and sinus osteitis, which were not evaluated via CT and endoscopy scores here. Type 2 inflammatory cytokines such as IL-5 and IL-13 are key mediators of airway inflammation in patients with CRS and asthma 2,3 and are thus correlated with these objective severity measurements of CT and endoscopy. Furthermore, MMPs such as MMP-1 and -9 may be predictors of  www.nature.com/scientificreports www.nature.com/scientificreports/ lower airway obstruction and resistance to contemporary steroid-based medical treatment in patients with CRS and asthma. Therefore, defining the role of MMPs and their association with immune pathways is imperative in designing, implementing, and tailoring therapeutics to successfully treat these patients. Future novel therapeutics www.nature.com/scientificreports www.nature.com/scientificreports/ that target MMP-related inflammatory responses may thus be beneficial to those with asthma or CRS that is refractory to current standard treatment protocols. For example, antibiotics such as doxycycline are established MMP-9 inhibitors may be beneficial to this group of patients.
While it offers potentially significant insights into the improved treatment of asthmatic patients with CRS, the present study has several limitations that warrant some consideration. First, this study would have benefited from the inclusion of patients without airway diseases and CRS in a normal control group. However, inclusion of such a group was not permitted by our institutional ethics committee due to the requirement for an invasive biopsy of the nasal mucosa. Second, a larger-scale study is necessary for further subgroup analyses, such as that of CRS patients with and without polyps or patient stratification by the severity of osteitis. Although scoring system of www.nature.com/scientificreports www.nature.com/scientificreports/ osteitis such as global osteitis scoring scale have been proposed 24 , we suggested it might be suitable for recurrent rather than our primary CRS cases. It not common for bony thickening >3 mm or >5 mm in primary CRS cases. We measured the maximal AE partition bone thickness on CT images to evaluate the severity of neo-osteogenesis instead and AE was selected because it is possible the most exposed sinus to the inhale cigarette smoke. Third, cigarette smoking data was collected by patient self-report, which is not always valid. Quantification of serum nicotine and cotinine levels and analysis of their relationships with cytokine expression should also be further considered to clarify the immune response associated with cigarette smoke exposure of the airway.  Maximal anterior ethmoid (AE) partition bone thickness measured on axial section sinus CT images was greater in smokers with CRS and asthma than in non-smokers with CRS and asthma, and asthmatic nonsmokers without CRS (a). MMP-1 (b) and MMP-9 (c) protein levels were correlated with maximal AE partition bone thickness on CT scans. However, CT scores were not correlated with maximal AE bone thickness (d). The number and significance are indicated.