Background: Chronic rhinosinusitis (CRS) is a heterogeneous chronic inflammatory disease generally divided based on the presence or absence of nasal polyps (NPs). One of the features of NPs is excessive fibrin deposition, which is associated with down-regulation of tissue plasminogen activator (t-PA) in NPs. As t-PA is expressed in epithelial cells, and epithelium is readily accessible to topical therapies, identifying compounds that can mediate the induction of t-PA would be a potential new strategy for the treatment of NPs. Objective: The objective of this study was to determine whether short-chain fatty acids (SCFAs) can induce t-PA in airway epithelial cells via their known receptors GPR41 and GPR43. Methods: We performed immunohistochemistry (IHC) to determine whether receptors for SCFAs, known as G protein-coupled receptor 41/free fatty acid receptor 3 (GPR41/FFAR3) and GPR43/FFAR2, are expressed in nasal tissue. Primary normal human bronchial epithelial (NHBE) cells were stimulated with different concentrations of SCFAs to test induction of t-PA, which was analysed by expression of mRNA and protein. Mediation of responses by SCFA receptors was evaluated by specific receptor gene silencing with siRNA. Results: Immunohistochemistry study revealed that airway epithelial cells expressed GPR41 and GPR43. Acetic acid, propionic acid, butyric acid and valeric acid significantly induced t-PA expression from two-to tenfolds. The strongest inducer of t-PA from NHBE cells was propionic acid; cells stimulated with propionic acid released t-PA into the supernatant in its active form. Gene silencing of GPR41 and GPR43 revealed that induction of t-PA by SCFAs was dependent upon both GPR41 and GPR43. Conclusions and Clinical Relevance: Short-chain fatty acids were shown to induce airway epithelial cell expression of t-PA via GPR41 and GPR43. Topical delivery of potent compounds that activate these receptors may have value by reducing fibrin deposition and shrinking nasal polyp growth.

BACKGROUND:B cells play many roles in health and disease. However, little is known about the mechanisms that drive B cell responses in the airways, especially in humans. Chronic rhinosinusitis (CRS) is an inflammatory disease of the upper airways that affects 10% of Europeans and Americans. A subset of CRS patients develop nasal polyps (NPs), which are characterized by type 2 inflammation, eosinophils and group 2 innate lymphoid cells (ILC2s). We have reported that NP contain elevated levels of B cells and antibodies, making NP an ideal system for studying B cells in the airways.OBJECTIVE:We sought to determine the mechanisms that drive B cell activation and antibody production during chronic airway inflammation.METHODS:We analysed B cells from NP or tonsil, or after ILC2 coculture, by flow cytometry. Antibody production from tissue was measured using Luminex assays and the frequency of antibody-secreting cells by ELISpot. Formation of B cell clusters was assessed using immunohistochemistry. Expression of genes associated with B cell activation and class switch recombination was measured by qRT-PCR.RESULTS:NP contained significantly elevated frequencies of plasmablasts, especially those that expressed the extrafollicular marker Epstein-Barr virus-induced protein 2 (EBI2), but significantly fewer germinal centre (GC) B cells compared with tonsil. Antibody production and the frequency of antibody-secreting cells were significantly elevated in NP, and there was evidence for local class switch recombination in NP. Finally, ILC2s directly induced EBI2 expression on B cells in vitro.CONCLUSIONS AND CLINICAL RELEVANCE:Our data suggest there is a unique B cell activation environment within NP that is distinct from classic GC-mediated mechanisms. We show for the first time that ILC2s directly induce EBI2 expression on B cells, indicating that ILC2s may play an important role in B cell responses. B cell-targeted therapies may provide new treatment options for CRSwNP.

BACKGROUND:Although chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by Th2 inflammation, the mechanism underlying the onset and amplification of this inflammation has not been fully elucidated. Dendritic cells (DCs) are major antigen-presenting cells, central inducers of adaptive immunity and critical regulators of many inflammatory diseases. However, the presence of DCs in CRS, especially in nasal polyps (NPs), has not been extensively studied.OBJECTIVE:The objective of this study was to characterize DC subsets in CRS.METHODS:We used real-time PCR to assess the expression of mRNA for markers of myeloid DCs (mDCs; CD1c), plasmacytoid DCs (pDCs; CD303) and Langerhans cells (LCs; CD1a, CD207) in uncinate tissue (UT) from controls and patients with CRS as well as in NP. We assayed the presence of DCs by immunohistochemistry and flow cytometry.RESULTS:Compared to UT from control subjects (n = 15) and patients with CRS without NP (CRSsNP) (n = 16) and CRSwNP (n = 17), mRNAs for CD1a and CD1c were significantly elevated in NPs (n = 29). In contrast, CD207 mRNA was not elevated in NPs. Immunohistochemistry showed that CD1c(+) cells but not CD303(+) cells were significantly elevated in NPs compared to control subjects or patients with CRSsNP. Flow cytometric analysis showed that CD1a(+) cells in NPs might be a subset of mDC1s and that CD45(+) CD19(-) CD1c(+) CD11c(+) CD141(-) CD303(-) HLA-DR(+) mDC1s and CD45(+) CD19(-) CD11c(+) CD1c(-) CD141(high) HLA-DR(+) mDC2s were significantly elevated in NPs compared to UT from controls and CRSsNP, but CD45(+) CD11c(-) CD303(+) HLA-DR(+) pDCs were only elevated in NPs compared to control UT.CONCLUSION AND CLINICAL RELEVANCE:Myeloid DCs are elevated in CRSwNP, especially in NPs. Myeloid DCs thus may indirectly contribute to the inflammation observed in CRSwNP.

Nasal mucosal innate host defense is essential for suppressing growth of pathogenic organisms and proper functioning of the upper airways. It has been recognized for long that sinonasal tissues express antimicrobial proteins important in host defense; however the regional distribution of these antimicrobial proteins in upper airways has not been studied in detail. In this study we analyzed the expression of selected antimicrobial proteins in sinonasal tissues at the front (inferior turbinate-IT) and rear (uncinate tissue - UT) of the anterior chamber of the nose in control subjects. Tissue samples were collected and analyzed by ELISA for expression of the antimicrobial proteins beta defensin-2 (hBD2), S100A7, SPLUNC1, lactoferrin, and pentraxin-3. Immunohistochemical analysis (IHC) was performed to further characterize the localization of two proteins (S100A7 and SPLUNC1) in sinonasal tissues samples. We observed a 4-21 fold regional differential expression of antimicrobial proteins in the sinonasal mucosa. S100A7 and hBD2 were more highly expressed in IT compared to UT, whereas lactoferrin and SPLUNC1 were more highly expressed in UT compared to IT. Interestingly, expression levels of pentraxin-3 were similar in IT and UT. We confirmed the differential localization of S100A7 and SPLUNC1 by IHC analysis. S100A7 was primarily expressed in mucosal epithelium and glands in IT. SPLUNC1 was highly expressed primarily in UT and was mostly glandular with minimal staining in mucosal epithelium. Our findings indicate the existence of marked regional variability in the expression of antimicrobial proteins in sinonasal tissues suggesting specialized regional functions of these proteins within the sinonasal cavity.

BACKGROUND:Chronic rhinosinusitis (CRS) is a disease characterized by inflammation of the nasal mucosa and paranasal sinuses. This inflammation may result in part from decreased epithelial barrier and innate immune responses, leading to frequent bacterial and fungal colonization. The objectives of this study were to investigate the expression of innate immune proteins of the palate lung and nasal epithelium clone (PLUNC) family in patients with CRS.METHODS:Nasal tissue samples were collected from control subjects and CRS patients with and without nasal polyps. Expression of the members of the PLUNC family was analyzed by real-time PCR. Expression of SPLUNC1 and LPLUNC2 proteins was analyzed by ELISA, immunoblot, and immunohistochemical analysis.RESULTS:Levels of mRNA for most of the members of the PLUNC family were profoundly reduced in nasal polyps (NPs) compared to uncinate tissue from control subjects or patients with CRS. LPLUNC2 and SPLUNC1 proteins were decreased in NPs of patients with CRS compared to uncinate tissue from control subjects. Immunohistochemical data revealed that within submucosal glands of sinonasal tissues, SPLUNC1 and LPLUNC2 were differentially expressed, in serous and mucous cells, respectively. The decrease in the expression of these molecules is probably explained by a decrease in the number of glands in NPs as revealed by correlations with levels of the glandular marker lactoferrin.CONCLUSIONS:Decreased SPLUNC1 and LPLUNC2 in NPs reflect a profound decrease in the number of submucosal glands. Decreased glands may lead to a localized defect in the production and release of glandular innate defense molecules.

RATIONALE: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with Th2-dominant inflammation including eosinophilia, in contrast to non-polypoid CRS (CRSsNP). CCL18/PARC is a CC chemokine known to recruit naïve T cells, B cells, and immature dendritic cells, and thought to be involved in Th2-related inflammatory diseases including asthma and atopic dermatitis. However, expression of CCL18 in CRS has not been extensively studied.METHODS: Using nasal polyp tissue (NP) and uncinate tissue (UT) from controls and patients with CRS, we examined the expression of CCL18 mRNA by real-time PCR and assayed CCL18 protein by ELISA, immunohistochemistry and western blot.RESULTS: Compared to control subjects (n=9), CCL18 mRNA was significantly increased in NP (432-fold, p<0.001, n=26) and UT (109-fold, p=0.009, n=19) from patients with CRSwNP but not in UT from patients with CRSsNP (n=18). Similarly, CCL18 protein was elevated in NP (4.75±1.13 ng/mg, p<0.001, n=38) and UT (1.52±0.40 ng/mg, p=0.066, n=29) from CRSwNP compared to UT from CRSsNP (0.52±0.06 ng/mg, n=32) and control subjects (0.52±0.11 ng/mg, n=16) in sinonasal tissue. An 8 kDa protein corresponding to CCL18 was detected by western blot only in patients with CRSwNP. Finally, immunofluorescence data showed CCL18 co-localization with CD68 positive macrophages as well as with other un-identified inflammatory cells in NP.CONCLUSIONS: Patients with CRSwNP have elevated levels of CCL18 in NP and UT. The role of CCL18 in the pathogenesis of CRSwNP is worthy of further investigation. RATIONALE: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with Th2-dominant inflammation including eosinophilia, in contrast to non-polypoid CRS (CRSsNP). CCL18/PARC is a CC chemokine known to recruit naïve T cells, B cells, and immature dendritic cells, and thought to be involved in Th2-related inflammatory diseases including asthma and atopic dermatitis. However, expression of CCL18 in CRS has not been extensively studied. METHODS: Using nasal polyp tissue (NP) and uncinate tissue (UT) from controls and patients with CRS, we examined the expression of CCL18 mRNA by real-time PCR and assayed CCL18 protein by ELISA, immunohistochemistry and western blot. RESULTS: Compared to control subjects (n=9), CCL18 mRNA was significantly increased in NP (432-fold, p<0.001, n=26) and UT (109-fold, p=0.009, n=19) from patients with CRSwNP but not in UT from patients with CRSsNP (n=18). Similarly, CCL18 protein was elevated in NP (4.75±1.13 ng/mg, p<0.001, n=38) and UT (1.52±0.40 ng/mg, p=0.066, n=29) from CRSwNP compared to UT from CRSsNP (0.52±0.06 ng/mg, n=32) and control subjects (0.52±0.11 ng/mg, n=16) in sinonasal tissue. An 8 kDa protein corresponding to CCL18 was detected by western blot only in patients with CRSwNP. Finally, immunofluorescence data showed CCL18 co-localization with CD68 positive macrophages as well as with other un-identified inflammatory cells in NP. CONCLUSIONS: Patients with CRSwNP have elevated levels of CCL18 in NP and UT. The role of CCL18 in the pathogenesis of CRSwNP is worthy of further investigation.

RATIONALE: Plasminogen activator inhibitor-1 (PAI-1) increases with adiposity, and promotes airway remodeling in murine asthma models. We sought to evaluate whether PAI-1 levels were associated with worse lung function in asthmatic subjects, independent of body mass index (BMI). METHODS: We measured plasma levels of PAI-1 by ELISA in 175 randomly selected adults with persistent asthma (aged 18-40y, female=141[81%]) from a community-based Chicago cohort. Age, self reported ethnicity, smoking status, and use of inhaled corticosteroid were obtained by questionnaire. Spirometry was performed according to ATS criteria. BMI was categorized into tertiles. Linear regression assessed the association between PAI-1 and FEV1%-predicted (Hankinson) and FVC %-predicted, controlling for age, gender, race/ethnicity, smoking status, and inhaled corticosteroid use. RESULTS: The median (IQR) for the PAI-1 level was 56.3 ng/ml (12.9-102.5), for FEV1 was 89.8% (76.0-99.3) and for FVC 99.7% (88.4-110.5). In adjusted analyses, PAI-1 was inversely correlated with FVC (β±SE=-0.12±0.05, p=0.02), and non-significantly so with FEV1 (β±SE=-0.07±0.06, p=0.25). The highest tertile of BMI was also inversely associated with FVC (β±SE=-7.98±3.16, p=0.01). The inclusion of BMI did not significantly change the association of PAI-1 with FVC (β±SE=-0.11±0.05, p=0.03). There was also an interaction between BMI tertiles and PAI-1 level on FVC (p=0.02 for second tertile). CONCLUSION: Plasma PAI-1 levels were inversely correlated with lung function in asthmatic subjects. This association was independent of the effects of other confounders such as BMI, suggesting an important role for PAI-1 in airway remodeling in subjects with asthma.

RATIONALE: Chronic rhinosinusitis (CRS) is a disease characterized by inflammation of the nasal mucosa and paranasal sinuses. This inflammation may result from decreased epithelial barrier and innate immune responses leading to frequent bacterial and fungal colonization. We tested whether CRS is associated with decreased expression of innate immune antimicrobial proteins of the Palate Lung Nasal Clone (PLUNC) family. METHODS: Nasal tissue and nasal lavage fluids were collected from control subjects and patients with CRS. Expression of long PLUNC2 (LPLUNC2) and short PLUNC1 (SPLUNC1) were analyzed by quantitative Real-time PCR. Expression of SPLUNC1 protein was also analyzed by immunoblot and immunohistochemical analysis. RESULTS: Levels of mRNA for LPLUNC2 (30 fold, p < 0.0002) and SPLUNC1 (15 fold, p < 0.003) were reduced in nasal polyps compared to uncinate tissue from control subjects. Expression of LPLUNC2 (4 fold, p < 0.014) and SPLUNC1 (5 fold, p = 0.051) proteins was also decreased in polyps compared to uncinate tissue from control patients but levels were not different in nasal lavage fluids of patients with CRS with nasal polyps compared to healthy controls. Immunohistochemical data revealed that SPLUNC1 was highly localized to the submucosal glands of uncinate and polyp tissue. There was a decreased glandular SPLUNC1 expression in polyps of patients with CRS compared to uncinate tissue from control subjects. CONCLUSIONS: Decreased SPLUNC1 in polyps may reflect a combination of reduced numbers of glands in polyps and reduced SPLUNC1 expression in glands residing within polyps. Lower SPLUNC1 expression in polyps may be caused by nasal polyp formation.

RATIONALE: Chronic rhinosinusitis (CRS) can be clinically classified into polypoid CRS (CRSwNP) and non-polypoid CRS (CRSsNP). Although CRSwNP and CRSsNP share several important clinical features, CRSwNP has more Th2-related inflammation. TSLP is a cytokine that triggers dendritic cell-mediated activation of Th2 inflammatory responses. However expression of TSLP in CRS has not been studied. METHODS: We investigated the expression of TSLP in primary human nasal epithelial cells (PNEC), and in nasal tissue and nasal lavage from control subjects and patients with CRS; we assayed the mRNA expression of TSLP and TSLPR using real-time PCR and assayed TSLP protein using specific ELISA. RESULTS: TSLP mRNA was significantly induced by IL-4 (2-fold) and dsRNA (26-fold) and the combination of IL-4 and dsRNA synergistically enhanced TSLP expression (62-fold) in PNEC. We assessed the expression of TSLP and TSLPR in nasal tissue. Although mRNA expression of TSLP and TSLPR was significantly increased in polyp tissue from patients with CRSwNP (TSLP; 2-fold, TSLPR; 3-fold, p < 0.05) compared to turbinate tissue from patients with CRS or healthy subjects, we did not find significant differences in the levels of TSLP protein in the tissue extracts (control;196 ± 39 pg/mg, n = 10, CRSsNP; 212 ± 51 pg/mg, n = 10, CRSwNP polyp; 91 ± 23 pg/mg, n = 10) or in nasal lavage (control; 133 ± 95 pg/ml, n = 6, CRSsNP; 67 ± 25 pg/ml, n = 12, CRSwNP; 51 ± 26 pg/ml, n = 6). CONCLUSIONS: Although nasal epithelial cells are capable of producing TSLP, levels of TSLP protein were not elevated in CRS and the pathogenic role of TSLP in CRS is therefore uncertain.

Chronic rhinosinusitis (CRS) can be clinically divided into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). Recent findings suggest plasma cells are increased in mucosae of patients with CRSwNP. BAFF and APRIL are cytokines that are essential in B cell and plasma cell responses. We report increased production of BAFF in primary human nasal epithelial cells (PNEC) activated in vitro and increased BAFF in sinonasal tissues and nasal lavage fluids derived from patients with CRSwNP. We investigated the expression of BAFF and APRIL in PNEC using real-time PCR. We also collected nasal tissue extracts and nasal lavage from healthy subjects and patients with CRS, and assayed BAFF protein using specific ELISA. In resting PNEC, expression of BAFF and APRIL was minimal. However, BAFF mRNA was significantly up-regulated by TLR3 ligand (dsRNA; 312-fold, p < 0.05), IFN-β (21-fold, p < 0.05) and IFN-γ (6-fold, p < 0.05). APRIL mRNA was weakly but significantly up-regulated by dsRNA (2-fold), IL-4 (2-fold), IFN-β (2-fold) and IFN-γ (2-fold) (p < 0.05 in all cases). We assessed the expression of BAFF and APRIL in nasal tissue from patients with CRS and controls. BAFF mRNA was significantly increased in patients with CRSwNP (p < 0.01) compared to patients with CRSsNP or healthy subjects. BAFF protein was also elevated in polypoid tissue and nasal lavage in patients with CRSwNP. Patients with CRSwNP have elevated levels of BAFF mRNA and protein in the nasal mucosa. The role of BAFF in the production of immunoglobulins by B cells in the epithelium of patients with CRSwNP is worthy of investigation.

Rationale The prevalence of asthma is increasing, especially among inner-city populations; aeroallergen sensitization may be a contributing factor. Our objective is to compare the prevalence of aeroallergen sensitization between low- and moderate-income, African American (AA) and non-AA asthmatics living in Chicago9s inner city. Methods The study sample is derived from the ongoing CHIRAH study: Chicago Health Initiative to Raise Asthma Health Equity. Participants are identified through the Chicago public and archdiocesan elementary schools. There were 37 middle-income (> $30,000 annual household income) and 52 low-income (< $30,000 annual household income) asthmatics, age 8 to 40 years, in whom specific aeroallergen IgE testing was performed: Alternaria, cat, cockroach, dog, dust mite, mouse, oak, ragweed, rat, and rye grass. Results Using Fisher9s exact statistics to compare the rate of allergen sensitization between low-income AAs and the other groups combined, a greater proportion of low-income AAs were sensitized to rat compared with other groups (p = .045). There was a trend for a greater proportion of low-income AAs to be sensitized to mouse (p = .057). Conclusion Sensitization to rat and mouse is more frequent in low-income AA asthmatics than in middle-income AAs or non-AAs, irrespective of income. Grant support: NHLBI UO1HL72478 and the Ernest S Bazley Trust.

RATIONALE: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with Th2-dominant inflammation including increased levels of IgE and eosinophilia, in contrast to CRS without nasal polyps (CRSsNP) which more resembles a Th1-related disease. BAFF is a cytokine that is essential in B cell responses. We report increased expression of BAFF in nasal epithelial cells activated in vitro and increased BAFF in mucosae of patients with CRSwNP. METHODS: We investigated the expression of BAFF in primary human nasal epithelial cells (PNEC) using real-time PCR. We also collected nasal tissue extracts and nasal lavage from healthy subjects and patients with CRSsNP and CRSwNP and assayed BAFF protein using a specific ELISA. RESULTS: In resting PNEC, expression of BAFF was minimal. However, BAFF mRNA was significantly up-regulated by TLR3 ligand (dsRNA; 406-fold, n=5, p<0.05) but not by TLR2 ligand (zymosan) or TLR4 ligand (LPS). We assessed the expression of BAFF in nasal tissue extracts and nasal lavage from patients with CRS and controls. In nasal tissue extracts, BAFF protein was significantly increased in patients with CRSwNP (25.6 pg/mg protein, p<0.02) compared to patients with CRSsNP (1.3 pg/mg protein) or healthy subjects (1.1 pg/mg protein). In addition, in nasal lavage, BAFF protein was only detected in patients with CRSwNP (4/8) but not in healthy subjects (0/7) or CRSsNP (0/5). CONCLUSIONS: Patients with polypoid CRS have elevated levels of BAFF protein in the nasal mucosa. The role of epithelial BAFF in the expression and class switching of immunoglobulins by B cells in CRSwNP is worthy of investigation.

RATIONALE: Since dust mite and mold extracts contain proteases that may degrade grass pollen, some allergists recommend administering grass extract separately from dust mite and mold extracts during immunotherapy. A few in vitro studies have demonstrated this degradation, however the clinical significance of this is unknown. Our study utilizes skin testing to evaluate whether there is a clinical difference in potency using combinations of grass with dust mite or mold compared to grass extract alone. METHODS: Dust mite and mold extracts were combined with grass extract and stored at 4°C for 0 and 12 weeks. Skin prick testing to various dilutions of combined grass extract with mold or dust and grass alone was performed on seven patients who were skin prick test positive to grass and skin prick and intradermal skin test negative to mold and dust mite. Erythema size, determined by adding the greatest diameter of flare to its perpendicular, was then compared for combination extracts versus grass alone. RESULTS: The mean change in erythema size from zero weeks to twelve weeks for a 1:20 dilution of grass extract alone was 14.43 mm whereas for dust mite and grass it was 13.00mm (p=0.797) and for mold and grass it was 9.75mm (p=0.453). CONCLUSION: Our data suggests the immunogenicity of grass allergen is unaffected by storage with dust mite or mold after three months. Studies with a larger number of subjects need to be performed to further elucidate this trend.

RATIONALE: The prevalence of asthma is increasing, especially among inner-city populations, and there is evidence that allergen sensitization is a contributing factor. Our objective is to characterize and contrast the prevalence of aeroallergen sensitization between children and adult asthmatics living in the inner city of Chicago. METHODS: The study sample was derived from the ongoing CHIRAH study: Chicago Initiative to Raise Asthma Health Equity. A screening questionnaire identified children and adults in their household with asthma. Fifty-six children and 33 adults participated. Plasma samples were tested for specific IgE antibodies for each of the 10 aeroallergens: alternaria, cat, cockroach, dog, dust mite, mouse, oak, ragweed, rat and rye grass. RESULTS: The prevalence of sensitization to at least one allergen was 80% (45/56) among children and 88% (29/33) among adults. Ragweed and cat were the most prevalent allergens in children (46.4%) and adults (66.7%), respectively. Using Chi-square to compare the rate of allergen sensitization between children and adults with asthma, there was no difference for each aeroallergen except cat (66.7% adults vs. 42.9% children, p=0.05). There was no significant difference in the rate of sensitization for indoor versus outdoor allergens between asthmatic children and adults. CONCLUSIONS: Both inner-city children and adults with asthma demonstrate a high degree of allergen sensitization. Based on these findings, the contribution of atopy is probably an equally significant factor in both inner-city children and adults with asthma.