Julie A. Poposki

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.

Regulation and Function of the IL-1 Family Cytokine IL-1F9 in Human Bronchial Epithelial Cells

The IL-1 family of cytokines, which now includes 11 members, is well known to participate in inflammation. Although the most recently recognized IL-1 family cytokines (IL-1F5-11) have been shown to be expressed in airway epithelial cells, the regulation of their expression and function in the epithelium has not been extensively studied. We investigated the regulation of IL-1F5-11 in primary normal human bronchial epithelial cells. Messenger (m)RNAs for IL-1F6 and IL-1F9, but not IL-1F5, IL-1F8 or IL-1F10, were significantly up-regulated by TNF, IL-1β, IL-17 and the Toll-like receptor (TLR)3 ligand double-stranded (ds)RNA. mRNAs for IL-1F7 and IL-1F11 (IL-33) were weakly up-regulated by some of the cytokines tested. Notably, mRNAs for IL-1F6 and IL-1F9 were synergistically enhanced by the combination of TNF/IL-17 or dsRNA/IL-17. IL-1F9 protein was detected in the supernatant following stimulation with dsRNA or a combination of dsRNA and IL-17. IL-1F6 protein was detected in the cell lysate but was not detected in the supernatant. We screened for the receptor for IL-1F9 and found that lung fibroblasts expressed this receptor. We found that IL-1F9 activated mitogen-activated protein kinases and the transcription factor NF-κB in primary normal human lung fibroblasts. IL-1F9 also stimulated the expression of the neutrophil chemokines IL-8 and CXCL3 and the Th17 chemokine CCL20 in lung fibroblasts. These results suggest that epithelial activation by TLR3 (e.g., by respiratory viral infection) and exposure to cytokines from Th17 cells (IL-17) and inflammatory cells (TNF) may amplify neutrophilic inflammation in the airway via induction of IL-1F9 and activation of fibroblasts.

Airway epithelial cells activate TH2 cytokine production in mast cells through IL-1 and thymic stromal lymphopoietin

BACKGROUND Airway epithelial cells are important regulators of innate and adaptive immunity. Although mast cells are known to play a central role in manifestations of allergic inflammation and are found in the epithelium in patients with T(H)2-related diseases, their role is incompletely understood. OBJECTIVES The objective of this study was to investigate the role of airway epithelial cells in the production of T(H)2 cytokines in mast cells. METHODS Normal human bronchial epithelial (NHBE) cells were stimulated with TNF, IL-4, IFN-γ, IL-17A, and double-stranded RNA (dsRNA) alone or in combination. Human mast cells were stimulated with epithelial cell-derived supernatants or cocultured with NHBE cells. T(H)2 cytokine responses were blocked with neutralizing antibodies. RESULTS Supernatants from IL-4- and dsRNA-stimulated NHBE cells significantly enhanced T(H)2 cytokine production from mast cells. The combination of IL-4 and dsRNA itself or supernatants from NHBE cells stimulated with other cytokines did not activate mast cells, suggesting that mast cell responses were induced by epithelial cell factors that were only induced by IL-4 and dsRNA. Epithelial supernatant-dependent T(H)2 cytokine production in mast cells was suppressed by anti-IL-1 and anti- thymic stromal lymphopoietin (TSLP) and was enhanced by anti-IL-1 receptor antagonist. Similar results were observed in coculture experiments. Finally, we found dsRNA-dependent production of IL-1, TSLP, and IL-1 receptor antagonist in NHBE cells was regulated by T(H) cytokines, and their ratio in NHBE cells correlated with T(H)2 cytokine production in mast cells. CONCLUSIONS Pathogens producing dsRNA, such as respiratory viral infections, might amplify local T(H)2 inflammation in asthmatic patients through the production of TSLP and IL-1 by epithelial cells and subsequent activation of T(H)2 cytokine production by mast cells in the airways.

Heterogeneous inflammatory patterns in chronic rhinosinusitis without nasal polyps in Chicago, Illinois

CRSsNP is a heterogenous disease but type 2 inflammation in CRSsNP was more common than type 1 inflammation among patients in Chicago, Illinois. Distinct therapeutic strategies may be needed depending on the type of inflammation found in CRSsNP.

Group 2 innate lymphoid cells are elevated and activated in chronic rhinosinusitis with nasal polyps

Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is characterized by type 2 inflammation with high levels of Th2 cytokines. Although T helper cytokines are released from T cells, innate lymphoid cells (ILC) are also known to produce high levels of the same cytokines. However, the presence of various types of ILC in CRS is poorly understood.

Elevated presence of myeloid dendritic cells in nasal polyps of patients with chronic rhinosinusitis.

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.

Chronic Airway Inflammation Provides a Unique Environment for B Cell Activation and Antibody Production

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.

Proprotein convertases generate a highly functional heterodimeric form of thymic stromal lymphopoietin in humans

Rationale: Thymic stromal lymphopoietin (TSLP) is known to be elevated and truncated in nasal polyps (NPs) of patients with chronic rhinosinusitis and might play a significant role in type 2 inflammation in this disease. However, neither the structure nor the role of the truncated products of TSLP has been studied. Objective: We sought to investigate the mechanisms of truncation of TSLP in NPs and the function of the truncated products. Methods: We incubated recombinant human TSLP with NP extracts, and determined the protein sequence of the truncated forms of TSLP using Edman protein sequencing and matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry. We investigated the functional activity of truncated TSLP using a PBMC‐based bioassay. Results: Edman sequencing and mass spectrometry results indicated that NP extracts generated 2 major truncated products, TSLP (residues 29–124) and TSLP (131–159). Interestingly, these 2 products remained linked with disulfide bonds and presented as a dimerized form, TSLP (29–124 + 131–159). We identified that members of the proprotein convertase were rate‐limiting enzymes in the truncation of TSLP between residues 130 and 131 and generated a heterodimeric unstable metabolite TSLP (29–130 + 131–159). Carboxypeptidase N immediately digested 6 amino acids from the C terminus of the longer subunit of TSLP to generate a stable dimerized form, TSLP (29–124 + 131–159), in NPs. These truncations were homeostatic but primate‐specific events. A metabolite TSLP (29–130 + 131–159) strongly activated myeloid dendritic cells and group 2 innate lymphoid cells compared with mature TSLP. Conclusions: Posttranslational modifications control the functional activity of TSLP in humans and overproduction of TSLP may be a key trigger for the amplification of type 2 inflammation in diseases.