Florian Sachse

Staphylococcus aureus invades the epithelium in nasal polyposis and induces IL‐6 in nasal epithelial cells in vitro

To cite this article: Sachse F, Becker K, von Eiff C, Metze D, Rudack C. Staphylococcus aureus invades the epithelium in nasal polyposis and induces IL‐6 in nasal epithelial cells in vitro. Allergy 2010; 65: 1430–1437.

Evaluation of treatment results with regard to initial anterior commissure involvement in early glottic carcinoma treated by external partial surgery or transoral laser microresection

Modalities of surgical treatment of early glottic carcinoma include transoral laser microresection and external partial surgery.

PAR‐2 activation regulates IL‐8 and GRO‐α synthesis by NF‐κB, but not RANTES, IL‐6, eotaxin or TARC expression in nasal epithelium

Background The effects of protease‐activated receptor‐2 (PAR‐2) stimulation on inflammation mechanisms of chronic rhinosinusitis (CRS) are still unknown.

Induction of CXC chemokines in A549 airway epithelial cells by trypsin and staphylococcal proteases − a possible route for neutrophilic inflammation in chronic rhinosinusitis

While various microorganisms have been recovered from patients with chronic rhinosinusitis, the inflammatory impact of virulence factors, in particular proteases from Staphylococcus aureus and coagulase negative staphylococci on the nasal epithelium, has not yet been investigated. Expression of CXC chemokines was determined in the epithelium of patients with chronic rhinosinusitis by immunohistochemistry. In a cell culture system of A549 respiratory epithelial cells, chemokine levels were quantified by enzyme‐linked immunosorbent assay (ELISA) after stimulation with supernatants originating from three different staphylococcal strains or with trypsin, representing a serine protease. Inhibition experiments were performed with prednisolone, with the serine protease inhibitor 4‐(2‐aminoethyl)‐benzenesulphonylfluoride (AEBSF) and with the nuclear transcription factor (NF)‐κΒ inhibitor (2E)‐3‐[[4‐(1,1‐dimethylethyl)phenyl]sulphonyl]‐2‐propenenitrite (BAY) 11–7085. Electromobility shift assays (EMSA) were used to demonstrate NF‐κB‐dependent protein synthesis. CXC chemokines interleukin (IL)‐8, growth‐related oncogene alpha (GRO‐α) and granulocyte chemotactic protein‐2 (GCP‐2) were expressed in the patients’ epithelium whereas epithelial cell‐derived neutrophil attractant 78 (ENA‐78) was rarely detected. In A549 cells, chemokines IL‐8, ENA‐78 and GRO‐α but not GCP‐2 were induced by trypsin and almost equal levels were induced by staphylococcal supernatants. IL‐8, GRO‐α and ENA‐78 synthesis was suppressed almost completely by AEBSF and BAY 11–7085, whereas prednisolone reduced chemokine levels differentially dependent on the supernatant added. CXC chemokines were detectable in the epithelium of patients with chronic rhinosinusitis. Staphylococcal serine proteases induced CXC chemokines in A549 cells, probably by the activation of proteases activated receptors, and thus might potentially be involved in neutrophilic inflammation in chronic sinusitis.

Promoter methylation of cyclin A1 is associated with human papillomavirus 16 induced head and neck squamous cell carcinoma independently of p53 mutation

Aberrant promoter methylation of specific genes and infection with human papillomavirus 16 (HPV16) are known risk factors for the development of Head and Neck Squamous Cell Carcinoma (HNSCC). Little knowledge exists on the interaction of HPV16 infection and promoter methylation in HNSCC. The promoter methylation status of 12 genes (TIMP3, CDH1, CDKN2A, DAPK1, transcription factor 21 (TCF21), CD44, MLH1, MGMT, RASSF1, cyclin A1 (CCNA1), LARS2, and CEBPA) was evaluated by methylation‐specific polymerase chain reaction in 55 primary HNSCC and 31 controls. The results were correlated with HPV16 status and clinicopathological characteristics. CCNA1 and p53 protein expression were additionally determined by immunohistochemistry and compared with p53 mutation status. Methylation of DAPK1 (P = 0.043), CCNA1 (P = 0.016) and TCF21 (P = 0.0005) was significantly more present in HNSCC than in controls. The genes TIMP3 (P = 0.018) and CCNA1 (P = 0.015) showed higher methylation frequency in HPV16 positive HNSCC compared to HPV16 negative tumors. CCNA1 methylation did not correlate with CCNA1 protein expression and p53 mutation, respectively. Methylation of TCF21 was associated with higher age (P = 0.044) and nicotine abuse (P = 0.035). Methylation of CCNA1 was significantly more present in females (P = 0.003). Methylation of TCF21 and CCNA1 are important risk factors for HNSCC development. CCNA1 methylation may play a crucial role in HPV16‐induced carcinogenesis of HNSCC independently of p53.

Neutrophil chemokines in epithelial inflammatory processes of human tonsils

CXC chemokines are thought to play an important role at sites of inflammation. Because ELR+ CXC chemokines are expressed in different types of tonsillitis we investigated the role of the surface/crypt epithelium of human tonsils  in  producing  ELR+  CXC  chemokines:  interleukin  (IL)‐8  (CXCL8), ENA‐78 (CXCL5), GRO‐α (CXCL1) and GCP‐2 (CXCL6). Tonsillar tissue was obtained from patients undergoing tonsillectomy and chemokine expression was investigated by means of immunohistochemistry. A549 cells were used as a model to study kinetics of chemokine expression in epithelial cells. Cells were stimulated with tumour necrosis factor (TNF)‐α, lipopolysaccharide (LPS) and supernatants derived from aerobic/anaerobic Staphylococcus aureus strains. Chemokine expression was measured by quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) and enzyme‐linked immunosorbent assay (ELISA). We observed epithelial expression of IL‐8, GRO‐α and GCP‐2 in different types of tonsillitis, whereas ENA‐78 was rarely expressed. In A549 cells abundant expression of ENA‐78 was detected. IL‐8 and GCP‐2 are expressed in an acute type of tonsillitis whereas GRO‐α was frequently detectable both in chronically and acutely inflamed tonsils. ENA‐78 does not seem to play a pivotal role in tonsillitis in vivo.

Incidence of staphylococcal colonization and of the 753Q Toll-like receptor 2 variant in nasal polyposis.

Background The impact of Staphylococcus aureus on the development of chronic sinusitis with nasal polyps (nasal polyposis [NP]) is a controversial discussion because different S. aureus colonization rates have been reported. Aside from the presence of a microbial stimulus, elements of innate immunity such as Toll-like receptors (TLRs) and/or impaired TLR function could be relevant for the development of this disease. Because the 753Q TLR2 variant may predispose to staphylococcal infection, we simultaneously analyzed staphylococcal colonization and the R753Q TLR2 single nucleotide polymorphism (SNP) in NP. Methods Sixty-eight patients with NP (47 men and 21 women; mean age [±SD], 51.8 years [16.3]) and 51 controls (32 men and 19 women; mean age [±SD], 36.3 years [12.2]) were included. Patient characteristics studied included status of allergy, asthma, aspirin intolerance, and endoscopic and CT polyp score. For detection of bacteria, standard procedures of bacteriology and 16S rRNA gene sequencing were used. The R753Q TLR2 polymorphism was studied by allelic discrimination assay. Results Overall, 128 isolates were cultured from 68 NP specimens, with Staphylococcus epidermidis and S. aureus being the most frequent bacterial isolates. Other bacterial species were infrequently detected. Fifty-nine isolates were cultured from 51 controls. Similarly, S. epidermidis and S. aureus were the most frequent bacterial isolates. S. aureus colonization was significantly increased in NP (p < 0.05). However, SNP genotyping results showed no association of the 753Q TLR2 variant with NP. Conclusions Although S. aureus detection was increased in NP, nasal polyp pathology is not related to the 753Q TLR2 variant.

Immunomodulation of Nasal Epithelial Cells by Staphylococcus aureus-Derived Serine Proteases

The impact of Staphylococcus aureus in the pathogenesis of chronic rhinosinusitis is not well understood. Therefore, we investigated primary human nasal epithelial cell cultures for their ability to produce IL-8, growth-related oncogene-α, and IL-6 via stimulation with trypsin and culture supernatants of different S. aureus strains and phenotypes. Inhibition of cytokine synthesis was performed using a glucocorticoid, a serine protease inhibitor, and a cysteine protease inhibitor. Finally, signal transduction pathways were analyzed by quantifying phosphorylated forms of MAPKs (PI3K, ERK, and p38) and DNA-binding assays that quantified NF-κB and its inhibition using BAY11-7085. In vitro studies showed that the induction of IL-8, growth-related oncogene-α, and IL-6 by S. aureus culture supernatants was significantly inhibited by the serine protease inhibitor. In contrast, steroids and the cysteine protease inhibitor had little effect. Activation of NF-κB was observed after cell treatment with trypsin and bacterial supernatants, and was inhibited by BAY11-7085 and the serine protease inhibitor. S. aureus serine proteases were identified to modulate chemokine synthesis and activate NF-κB in nasal epithelial cells, and may therefore be relevant for the pathophysiology of chronic rhinosinusitis.

Proinflammatory Impact of Staphylococcus epidermidis on the Nasal Epithelium Quantified by IL-8 and GRO-α Responses in Primary Human Nasal Epithelial Cells

Background: Bacterial etiology of chronic rhinosinusitis (CRS) still remains controversial. Whereas Staphylococcus aureus enterotoxins have been detected in CRS, the impact of Staphylococcus epidermidis, a major commensal inhabitant of the nose, has not been studied. Among others, serine and cysteine proteases have been identified as factors of virulence in S. epidermidis. Methods:S. epidermidis was examined in tissue biopsies of 30 CRS patients (16 with nasal polyposis) using standard procedures. Primary human nasal epithelial cells from inferior nasal turbinates (HNECs), from nasal polyps (NPECs) and A549 airway epithelial cells were stimulated with S. epidermidis supernatants DSM20044 or ATCC35984 and the IL-8 and GRO-α response was quanti- fied by ELISA. Protease-triggered chemokine responses and involvement of NF-ĸB were investigated by addition of protease or NF-ĸB inhibitors. Activation of NF-ĸB was demonstrated by quantitative DNA binding assay. Results:S. epidermidis was the most frequently isolated bacteria in the majority of CRS patients. HNECs and NPECs revealed no different IL-6 and IL-8 synthesis following stimulation with DSM20044 or ATCC35984. Stimulation of HNECs and A549 cells with S. epidermidis supernatants resulted in increased IL-8 and GRO-α expression which could be suppressed by the serine protease inhibitor AEBSF and the NF-ĸB inhibitor BAY 11 but not by the cysteine protease inhibitor E64. Results obtained for A549 cells were similar to HNECs. Conclusion:S. epidermidis was present in the majority of CRS specimens. Proinflammatory impact of S. epidermidis supernatants on nasal epithelial cells was demonstrated by serine protease-triggered and NF-ĸB-dependent chemokine responses.

IKK-2 inhibitor TPCA-1 represses nasal epithelial inflammation in vitro.

BACKGROUND Nasal polyposis (NP) is considered a subgroup within chronic rhinosinusitis. NP can be further subdivided into aspirin sensitive- and aspirin tolerant types (ASNP/ ATNP). Although the true etiology of NP has not been identified so far, it is agreed that NP represents an inflammatory disease of the nasal mucosa. Alterations of cellular kinase activities including that of IKK-2 might play a role in this inflammatory process. METHODS Paraffin sections of ASNP, ATNP and controls were immunostained with Phospho-IkB-α antibody that detects the direct IKK-2 product (IkB-α. Intensity of epithelial staining was analysed semi-quantitatively by two independent observers. In cultured nasal polyp epithelial cells (NPECs) epithelial derived cytokines IL-8 and GRO α were induced by TNF-α or Staphylococcal supernatants and subsequently repressed by IKK-2 inhibitor TPCA-1. RESULTS Significant Phospho-IkB-α staining was observed in the nasal epithelium of ASNP compared to ATNP and controls suggesting strong IKK-2 activation in patients with ASNP in vivo. In vitro, pro-inflammatory cytokines IL-8 and GRO-α in NPECs were significantly repressed by TPCA-1. CONCLUSION IKK-2 activity is increased in the subgroup of ASNP. IL-8 and GRO-α responses were repressed by IKK-2 inhibitor TPCA-1 in vitro. IKK-2 inhibitors might represent a potential target for anti-inflammatory intervention in ASNP.