Kathryn E. Hulse

Thymic stromal lymphopoietin activity is increased in nasal polyps of patients with chronic rhinosinusitis

BACKGROUND Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with TH2-dominant inflammation. Thymic stromal lymphopoietin (TSLP) is a cytokine that triggers dendritic cell-mediated TH2 inflammatory responses and that enhances IL-1-dependent TH2 cytokine production in mast cells. Although increased TSLP mRNA levels have been found in nasal polyps (NPs), expression of TSLP protein and its function in patients with chronic rhinosinusitis (CRS) have not been fully explored. OBJECTIVES The objective of this study was to investigate the role of TSLP in patients with CRS. METHODS We investigated the presence and stability of TSLP protein in NPs using ELISA and Western blotting and investigated the function of TSLP in nasal tissue extracts with a bioassay based on activation of human mast cells. RESULTS Although TSLP mRNA levels were significantly increased in NP tissue from patients with CRSwNP compared with uncinate tissue from patients with CRS or control subjects, TSLP protein was significantly decreased in NP tissue, as detected by using the commercial ELISA kit. We found that recombinant TSLP was time-dependently degraded by NP extracts, and this degradation was completely inhibited by a protease inhibitor cocktail, suggesting that TSLP is sensitive to tissue proteases. Interestingly, NP extract-treated TSLP had higher activity in mast cells, although the amount of full-length TSLP was reduced up to 85%. NP extracts significantly enhanced IL-1β-dependent IL-5 production in mast cells compared with uncinate tissue homogenates, and responses were significantly inhibited by anti-TSLP, suggesting that NPs contain biologically relevant levels of TSLP activity. CONCLUSION TSLP and its metabolic products might play an important role in the inflammation seen in patients with CRSwNP.

Evidence for altered activity of the IL-6 pathway in chronic rhinosinusitis with nasal polyps

BACKGROUND IL-6 activates T(H)17 cells and regulates the response of B lymphocytes and regulatory T cells. The IL-6 receptor and the membrane protein, glycoprotein 130 (gp130), form an active signaling complex that signals through signal transducer and activator of transcription 3 (STAT3) and other signaling molecules. Both the IL-6 receptor (IL-6R) and gp130 can be found in soluble forms that regulate the pathway. OBJECTIVE We measured IL-6 signaling components and IL-17 in chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), CRS without nasal polyps (CRSsNP), and controls to assess the IL-6 pathway in CRS. METHODS IL-6, soluble IL-6R, soluble gp130 (sgp130), and IL-17 were measured in sinus tissue extracts and in nasal lavage fluid by either cytokine bead array or ELISA. phosphoSTAT3 (p-STAT3) was determined by Western blot and by immunohistochemistry. RESULTS IL-6 protein was significantly (P < .001) increased in CRSwNP compared with CRSsNP and controls. Soluble IL-6R was also increased in nasal polyp compared with control tissue (P < .01). Despite elevated IL-6 and sIL-6R, IL-17A, E, and F were undetectable in the sinus tissue from most of the patients with CRS and controls. p-STAT3 levels were reduced in the polyp tissue, possibly indicating reduced activity of IL-6 in the tissue. sgp130 was elevated in CRSwNP compared with CRSsNP and controls. CONCLUSION p-STAT3 levels are decreased in CRSwNP despite increased levels of IL-6 and sIL-6R and are associated with the absence of an IL-17 response. This may be a response to elevated levels of sgp130, a known inhibitor of IL-6 signaling. These results indicate that IL-6 and its signaling pathway may be altered in CRSwNP.

Cytokines in Chronic Rhinosinusitis. Role in Eosinophilia and Aspirin-exacerbated Respiratory Disease

RATIONALE The mechanisms that underlie the pathogenesis of chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), and aspirin-exacerbated respiratory disease (AERD) are not clear. OBJECTIVES To first evaluate the inflammatory profiles of CRSsNP and CRSwNP tissues and then to investigate whether clinical differences observed between CRSwNP and AERD are in part secondary to differences in inflammatory mediator expression within nasal polyp (NP) tissues. METHODS Expression levels of numerous inflammatory mediators were determined by quantitative real-time polymerase chain reaction, ELISA, and multiplex immunoassay. MEASUREMENTS AND MAIN RESULTS CRSwNP NP had increased levels of type 2 mediators, including IL-5 (P < 0.001), IL-13 (P < 0.001), eotaxin-2 (P < 0.001), and monocyte chemoattractant protein (MCP)-4 (P < 0.01), compared with sinonasal tissue from subjects with CRSsNP and control subjects. Expression of IFN-γ messenger RNA or protein was low and not different among the chronic rhinosinusitis subtypes examined. Compared with CRSwNP, AERD NP had elevated protein levels of eosinophil cationic protein (ECP) (P < 0.001), granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.01), and MCP-1 (P = 0.01), as well as decreased gene expression of tissue plasminogen activator (tPA) (P = 0.02). Despite the higher eosinophilia in AERD, there was no associated increase in type 2 mediator protein levels observed. CONCLUSIONS CRSwNP was characterized by a predominant type 2 inflammatory environment, whereas CRSsNP did not reflect a classic type 1 milieu, as has been suggested previously. AERD can be distinguished from CRSwNP by elevated ECP levels, but this enhanced eosinophilia is not associated with elevations in traditional type 2 inflammatory mediators associated with eosinophil proliferation and recruitment. However, other factors, including GM-CSF, MCP-1, and tPA, may be important contributors to AERD pathogenesis.

Chronic rhinosinusitis with nasal polyps is characterized by B-cell inflammation and EBV-induced protein 2 expression

BACKGROUND Despite the high prevalence and morbidity of chronic rhinosinusitis (CRS), little is known about the mechanisms that underlie its pathogenesis. Recent studies have suggested that B cells might play an important role in CRS. OBJECTIVE We sought to thoroughly characterize B lineage cells within sinus tissues of patients with CRS and healthy control subjects and to determine whether levels of EBV-induced protein 2, which is known to play an important role in the development of B-cell responses, were increased in patients with CRS. METHODS Cells isolated from sinus tissues of patients with CRS and healthy control subjects were characterized by means of flow cytometry and immunohistochemistry. Local production of antibodies was measured in tissue extracts, nasal lavage fluid, and sera by using multiplex bead arrays and ELISA. Quantitative RT-PCR, ELISA, and Western blotting were used to assess gene and protein expression from tissue extracts. RESULTS Nasal polyps (NPs) from patients with CRS had increased levels of both B cells and plasma cells compared with uncinate tissue from healthy control subjects (P<.05). NPs also contained significantly increased levels of several antibody isotypes compared with normal uncinate tissue (P<.05), but no differences in circulating antibody levels were found. Interestingly, levels of EBV-induced protein 2 were also increased in NPs (P<.05) and were positively correlated with expression of plasma cell markers (CD138 and B lymphocyte-induced maturation protein) in sinus tissue. CONCLUSION B cells and plasma cells are enriched in NPs, actively produce antibodies locally, and might contribute to chronic inflammation in patients with CRS. Elucidating the mechanisms that underlie this excessive local B-cell response might provide novel insights for the development of improved therapeutic strategies.

Pathogenesis of nasal polyposis

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a complex inflammatory condition that affects a large proportion of the population world‐wide and is associated with high cost of management and significant morbidity. Yet, there is a lack of population‐based epidemiologic studies using current definitions of CRSwNP, and the mechanisms that drive pathogenesis in this disease remain unclear. In this review, we summarize the current evidence for the plethora of factors that likely contribute to CRSwNP pathogenesis. Defects in the innate function of the airway epithelial barrier, including diminished expression of antimicrobial products and loss of barrier integrity, combined with colonization by fungi and bacteria likely play a critical role in the development of chronic inflammation in CRSwNP. This chronic inflammation is characterized by elevated expression of many key inflammatory cytokines and chemokines, including IL‐5, thymic stromal lymphopoietin and CCL11, that help to initiate and perpetuate this chronic inflammatory response. Together, these factors likely combine to drive the influx of a variety of immune cells, including eosinophils, mast cells, group 2 innate lymphoid cells and lymphocytes, which participate in the chronic inflammatory response within the nasal polyps. Importantly, however, future studies are needed to demonstrate the necessity and sufficiency of these potential drivers of disease in CRSwNP. In addition to the development of new tools and models to aid mechanistic studies, the field of CRSwNP research also needs the type of robust epidemiologic data that has served the asthma community so well. Given the high prevalence, costs and morbidity, there is a great need for continued research into CRS that could facilitate the development of novel therapeutic strategies to improve treatment for patients who suffer from this disease.

B-lymphocyte lineage cells and the respiratory system

Adaptive humoral immune responses in the airways are mediated by B cells and plasma cells that express highly evolved and specific receptors and produce immunoglobulins of most isotypes. In some cases, such as autoimmune diseases or inflammatory diseases caused by excessive exposure to foreign antigens, these same immune cells can cause disease by virtue of overly vigorous responses. This review discusses the generation, differentiation, signaling, activation, and recruitment pathways of B cells and plasma cells, with special emphasis on unique characteristics of subsets of these cells functioning within the respiratory system. The primary sensitization events that generate B cells responsible for effector responses throughout the airways usually occur in the upper airways, tonsils, and adenoid structures that make up the Waldeyer ring. On secondary exposure to antigen in the airways, antigen-processing dendritic cells migrate into secondary lymphoid organs, such as lymph nodes, that drain the upper and lower airways, and further B-cell expansion takes place at those sites. Antigen exposure in the upper or lower airways can also drive expansion of B-lineage cells in the airway mucosal tissue and lead to the formation of inducible lymphoid follicles or aggregates that can mediate local immunity or disease.

Excessive fibrin deposition in nasal polyps caused by fibrinolytic impairment through reduction of tissue plasminogen activator expression.

RATIONALE Nasal polyps (NPs) are characterized by intense edema or formation of pseudocysts filled with plasma proteins, mainly albumin. However, the mechanisms underlying NP retention of plasma proteins in their submucosa remain unclear. OBJECTIVES We hypothesized that formation of a fibrin mesh retains plasma proteins in NPs. We assessed the fibrin deposition and expression of the components of the fibrinolytic system in patients with chronic rhinosinusitis (CRS). METHODS We assessed fibrin deposition in nasal tissue from patients with CRS and control subjects by means of immunofluorescence. Fibrinolytic components, d-dimer, and plasminogen activators were measured using ELISA, real-time PCR, and immunohistochemistry. We also performed gene expression and protein quantification analysis in cultured airway epithelial cells. MEASUREMENTS AND MAIN RESULTS Immunofluorescence data showed profound fibrin deposition in NP compared with uncinate tissue (UT) from patients with CRS and control subjects. Levels of the cross-linked fibrin cleavage product protein, d-dimer, were significantly decreased in NP compared with UT from patients with CRS and control subjects, suggesting reduced fibrinolysis (P < 0.05). Expression levels of tissue plasminogen activator (t-PA) mRNA and protein were significantly decreased in NP compared with UT from patients with CRS and control subjects (P < 0.01). Immunohistochemistry demonstrated clear reduction of t-PA in NP, primarily in the epithelium and glands. Th2 cytokine-stimulated cultured airway epithelial cells showed down-regulation of t-PA, suggesting a potential Th2 mechanism in NP. CONCLUSIONS A Th2-mediated reduction of t-PA might lead to excessive fibrin deposition in the submucosa of NP, which might contribute to the tissue remodeling and pathogenesis of CRS with nasal polyps.

Basophils are elevated in nasal polyps of patients with chronic rhinosinusitis without aspirin sensitivity

To the Editor: Chronic rhinosinusitis (CRS) is a common chronic diseasewith significant burden and effect on the quality of life of affected individuals, characterized by inflammation of the nasal mucosa and paranasal sinuses. Patients can be subdivided into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). In addition, aspirin exacerbated respiratory disease (AERD) is a subgroup of CRSwNP characterized by the triad of CRSwNP, asthma, and hypersensitivity to aspirin or nonsteroidal antiinflammatory drugs. Eosinophilia is evident in the nasal mucosa and submucosa of nasal polyps (NPs) from patients in Europe and the United States, but less so in Asian countries such as China or Japan. In this study we attempted, for the first time, to evaluate the role of another important inflammatory leukocyte, the basophil, in CRS. Basophils are granulocytes found mainly in the circulation and are known to have a role in allergic diseases, parasite expulsion, and immunity against ectoparasites. Basophil numbers are elevated in the bronchial mucosa and submucosa of asthmatic patients, the nasal submucosa in patients with allergic rhinitis, and in the skin of those with multiple inflammatory dermatologic conditions, including eczema and urticaria. AlthoughCRSwNP is a highlyTH2-biaseddisease,making basophil involvement likely, there are no reports of basophils in CRS. In the current study we used 2D7, a mouse mAb against a basophilspecific intermediate form of major basic protein, to detect basophils in uncinate tissue (UT)or polyp tissue obtained during surgery from patients with CRS and control subjects as described previously. This antibody is shown to be a specific marker for basophils and does not recognize any other cell type. We used the immunohistochemistry protocol detailed in previous studies using 2D7. Mucosa and submucosa in the tissue biopsies were delineated, and the total number of positively stained cells in each field was counted in 50 random hpf with 1003magnification. Results are reported as the number of positive cells per square millimeter. A tissue section was stained with hematoxylin and eosin. Eosinophils were identified on the basis of their cellular features and distinct cytoplasmic eosinophilic granules. For all polyp samples, a serial section was stained with a tryptase mAB, a specificmarker for mast cells as described previously. Cells were counted and data expressed as described above for basophils. Data are presented as mean 6 SEM. Comparisons between groups were assessed by using ANOVA and Newman-Keuls multiple comparison tests. Correlations were measured using a Spearman rank correlation test. All statistical analyses were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, Calif; www.graphpad.com). A P value of less than .05 was considered statistically significant. A total of 73 sections were labeled with 2D7, including polyps from 27 patients with and without AERD (10 with AERD and 17 without AERD) and UT from 16 patients with CRSwNP, 15 patients with CRSsNP, and 15 control cases with no evidence of CRS. Cells were identified as basophils on the basis of a brown cytoplasmic granular staining pattern (Fig 1, A). Negative control slides were labeled with an isotype-matched nonimmune antibody and showed no staining. As detailed in Table I, there were no 2D7 basophils detected within the uncinate mucosa and submucosa of control subjects. A small number of these cells were detected in the UTof patients with CRSsNP, and a greater number was detected in the UTof patients with CRSwNP, though the quantities detected were not significantly greater than in control UT. The 2D7-positive cells per square millimeter of tissue were significantly higher in the NP tissue of patients with CRSwNP than in the UT of control subjects or patients with CRSsNP and CRSwNP (Fig 1, B). This count was also significantly higher when compared with the corresponding UT of the same patients with CRSwNP, paired sets of the polyp tissue and the UT having been evaluated from 12 patients with CRSwNP. Interestingly, the number of basophils detected in NPs from subjects with AERD was not higher than in normal or CRS UT and was significantly lower than in NPs from patients without AERD. Eosinophil counts in the UT of control patients with no evidence of sinusitis were significantly lower than in the UT of patients with CRSwNP (Table I). Polyp tissue had a significantly higher number of eosinophils than did the UT of all groups, including controls and patients with CRSsNP and CRSwNP. Polyps from subjects with AERD had a significantly higher eosinophil count than did the UT of all groups, with a trend toward higher levels than in non-AERD polyps. Basophil and eosinophil counts in all tissues (excluding AERD polyps) significantly correlated with one another (P < .001; Spearman r 5 0.67) (Fig 1, D). But the basophil and mast cell counts did not correlate. Basophils and eosinophils utilize similar pathways for recruitment and might be expected to be recruited to an inflammatory site in parallel.We therefore calculated the ratio of eosinophils to basophils by dividing the total number of eosinophils per hpf by the number of basophils per hpf in a fixed area in serially cut slides. The eosinophil to basophil ratio in normal UT had a mean value of 3.0; this value was 13.1 in CRSsNP UT and 49.0 in CRSwNPUT. Furthermore, the magnitude of the ratio of eosinophils to basophils was highest in NPs, with a mean of 89.2 in non-AERD polyps and 287.3 in AERD polyps. Values of this ratio were significantly higher in polyps than in the UT of CRSwNP, CRSsNP, and control cases. The blood basophil counts within 6 months of the time of surgery were available for 40 cases: 13 with analysis of polyp tissue and 27 with analysis of the UT. There was no correlation between the blood basophil count and the number of basophils in either the polyp tissue or the UT. This appears to exclude systemic basophilia as a likely cause of the observed increase in the number of basophils in the polyp tissue. We also correlated the basophil numbers in polyps with total serum IgE (available in 13 cases) and the presence of anosmia or hyposmia as a marker for disease severity and positive skin test result. None of these comparisons was significant (both including and excluding AERD polyps). Potentially other patient-related factors, such as treatment, might have affected the number of basophils. We compared the number of basophils in the UT and the polyp tissue of patients who were on oral steroids at the time of surgery compared with those who

Immune Mechanisms of Chronic Rhinosinusitis

Chronic rhinosinusitis (CRS) is a common inflammatory disease that results in a significant decrease in patient quality of life and a large economic burden. However, the lack of population-based epidemiologic studies and robust model systems has made it difficult to fully elucidate the key inflammatory pathways that drive the chronic inflammatory responses observed in CRS. This review will highlight the wide variety of factors that likely contribute to CRS disease pathogenesis. Defects in the innate immune function of the airway epithelium, including decreases in barrier function, mucociliary clearance, and production of antimicrobial peptides, all likely play a role in the initial inflammatory response. Subsequent recruitment and activation of eosinophils, mast cells, and innate lymphoid cells (ILCs) further contributes to the chronic inflammatory response and directly activates adaptive immune cells, including T and B cells. However, development of new tools and model systems is still needed to further understand the chronicity of this inflammatory response and which specific factors are necessary or sufficient to drive CRS pathogenesis. Such studies will be critical for the development of improved therapeutic strategies aimed at treating this highly prevalent and costly disease.

Increased expression of factor XIII-A in patients with chronic rhinosinusitis with nasal polyps

BACKGROUND Profound edema or formation of a pseudocyst containing plasma proteins is a prominent characteristic of nasal polyps (NP). However, the mechanisms underlying NP retention of plasma proteins in the submucosa remain unclear. Recently, we reported that impairment of fibrinolysis causes excessive fibrin deposition in NP and this might be involved in the retention of plasma proteins. Although the coagulation cascade plays a critical role in fibrin clot formation at extravascular sites, the expression and role of coagulation factors in NP remain unclear. OBJECTIVE The objective of this study was to investigate the expression of coagulation factors in patients with chronic rhinosinusitis (CRS). METHODS Sinonasal tissues were collected from patients with CRS and control subjects. We assayed mRNA for factor XIII-A (FXIII-A) by using real-time PCR and measured FXIII-A protein by means of ELISA, immunohistochemistry, and immunofluorescence. RESULTS FXIII-A mRNA levels were significantly increased in NP tissue from patients with CRS with NP (P < .001) compared with uncinate tissue from patients with CRS or control subjects. Similarly, FXIII-A protein levels were increased in NP. Immunofluorescence analysis revealed that FXIII-A expression in inflammatory cells and FXIII-A(+) cell numbers were significantly increased in NP. Most FXIII-A staining was observed within CD68(+)/CD163(+) M2 macrophages in NP. Levels of FXIII-A correlated with markers of M2 macrophages, suggesting that M2 macrophages are major FXIIIA-producing cells in NP. CONCLUSION Overproduction of FXIII-A by M2 macrophages might contribute to the excessive fibrin deposition in the submucosa of NP, which might contribute to the tissue remodeling and pathogenesis of CRS with NP.