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Pulmonary Mycosis Drives Forkhead Box Protein A2 Degradation and Mucus Hypersecretion through Activation of the Spleen Tyrosine Kinase–Epidermal Growth Factor Receptor–AKT/Extracellular Signal-Regulated Kinase 1/2 Signaling

2020; Elsevier BV; Volume: 191; Issue: 1 Linguagem: Inglês

10.1016/j.ajpath.2020.09.013

1525-2191

Woosuk Choi, Alina X. Yang, Aaron Sieve, Shanny Hsuan Kuo, Srinivasu Mudalagiriyappa, Miranda D. Vieson, Carol W. Maddox, Som G. Nanjappa, Gee W. Lau,

Infectious Diseases and Mycology

Pulmonary mycoses are difficult to treat and detrimental to patients. Fungal infections modulate the lung immune response, induce goblet cell hyperplasia and metaplasia, and mucus hypersecretion in the airways. Excessive mucus clogs small airways and reduces pulmonary function by decreasing oxygen exchange, leading to respiratory distress. The forkhead box protein A2 (FOXA2) is a transcription factor that regulates mucus homeostasis in the airways. However, little is known whether pulmonary mycosis modulates FOXA2 function. Herein, we investigated whether Blastomyces dermatitidis and Histoplasma capsulatum–infected canine and feline lungs and airway epithelial cells could serve as higher animal models to examine the relationships between fungal pneumonia and FOXA2-regulated airway mucus homeostasis. The results indicate that fungal infection down-regulated FOXA2 expression in airway epithelial cells, with concomitant overexpression of mucin 5AC (MUC5AC) and mucin 5B (MUC5B) mucins. Mechanistic studies reveal that B. dermatitidis infection, as well as β-glucan exposure, activated the Dectin-1–SYK–epidermal growth factor receptor–AKT/extracellular signal-regulated kinase 1/2 signaling pathway that inhibits the expression of FOXA2, resulting in overexpression of MUC5AC and MUC5B in canine airway cells. Further understanding of the role of FOXA2 in mucus hypersecretion may lead to novel therapeutics against excessive mucus in both human and veterinary patients with pulmonary mycosis. Pulmonary mycoses are difficult to treat and detrimental to patients. Fungal infections modulate the lung immune response, induce goblet cell hyperplasia and metaplasia, and mucus hypersecretion in the airways. Excessive mucus clogs small airways and reduces pulmonary function by decreasing oxygen exchange, leading to respiratory distress. The forkhead box protein A2 (FOXA2) is a transcription factor that regulates mucus homeostasis in the airways. However, little is known whether pulmonary mycosis modulates FOXA2 function. Herein, we investigated whether Blastomyces dermatitidis and Histoplasma capsulatum–infected canine and feline lungs and airway epithelial cells could serve as higher animal models to examine the relationships between fungal pneumonia and FOXA2-regulated airway mucus homeostasis. The results indicate that fungal infection down-regulated FOXA2 expression in airway epithelial cells, with concomitant overexpression of mucin 5AC (MUC5AC) and mucin 5B (MUC5B) mucins. Mechanistic studies reveal that B. dermatitidis infection, as well as β-glucan exposure, activated the Dectin-1–SYK–epidermal growth factor receptor–AKT/extracellular signal-regulated kinase 1/2 signaling pathway that inhibits the expression of FOXA2, resulting in overexpression of MUC5AC and MUC5B in canine airway cells. Further understanding of the role of FOXA2 in mucus hypersecretion may lead to novel therapeutics against excessive mucus in both human and veterinary patients with pulmonary mycosis. Dimorphic fungi, including Blastomyces dermatitidis, Histoplasma capsulatum, Coccidiodes immitis, and Coccidiodes posadasii, are important etiologic pathogens of pulmonary mycoses. B. dermatitidis is present in the soil and wet decaying wood of eastern North America and central United States.1Bariola J. Vyas K. Pulmonary blastomycosis.Semin Respir Crit Care Med. 2011; 32: 745-753Crossref PubMed Scopus (20) Google Scholar, 2McBride J.A. Gauthier G.M. Klein B.S. Clinical manifestations and treatment of blastomycosis.Clin Chest Med. 2017; 38: 435-449Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 3Smith J. Gauthier G. 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An official American Thoracic Society statement: treatment of fungal infections in adult pulmonary and critical care patients.Am J Respir Crit Care Med. 2011; 183: 96-128Crossref PubMed Scopus (365) Google Scholar,49Arceneaux K. Taboada J. Hosgood G. Blastomycosis in dogs: 115 cases (1980-1995).J Am Vet Med Assoc. 1998; 213: 658-664PubMed Google Scholar However, it is unclear whether fungal infections modulate FOXA2 function. In this study, we explored whether B. dermatitidis and H. capsulatum–infected canine and feline lungs and airway cells can serve as higher animal models to examine FOXA2 function during fungal pneumonia. Unless stated otherwise, chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO). The Saccharomyces cerevisiae β-glucan (CAS 9012 to 72-0; EMD Millipore, Burlington, MA) is a major structural element of fungal cell wall composed of linear β1,3-glucan with a small number of β1,6-glucan branches. Antibodies were used at dilutions recommended by manufacturers and optimized as needed. Antibodies were diluted in 5% bovine serum albumin. Antibodies against FOXA2 [sc-10160; immunohistochemistry (IHC) 1:250; Western blot 1:1000], MUC5AC (sc-20118; IHC 1:250; Western blot 1:1000), MUC5B (sc-20119; IHC 1:500; Western blot 1:1000), STAT6 (sc-1689; Western blot 1:1000), phosphorylated STAT6 (sc-71793; Western blot 1:1000), EGFR (sc-03; Western blot 1:1000), phosphorylated EGFR (pEGFR; sc-101668; Western blot 1:1000), AKT (Western blot 1:1000), phosphorylated AKT (pAKT; sc-16646; Western blot 1:1000), and glyceraldehyde-3-phosphate dehydrogenase (sc-166545; Western blot 1:1000) were purchased from Santa Cruz Biotechnology (Dallas, TX). Antibodies against SYK (13,198; Western blot 1:1000), phosphorylated SYK (2710; Western blot 1:1000), extracellular signal-regulated kinase (ERK) 1/2 (4695; Western blot 1:1000), phosphorylated ERK1/2 (pERK1/2; 4370; Western blot 1:1000), and histone H3 (9717; Western blot 1:1000) were purchased from Cell Signaling Technology (Danvers, MA). Specific inhibitors AG1478 (658,552), LY294002 (440,202), and PD98059 (513,000) were purchased from Sigma Aldrich. Piceatannol (SC-200610) was purchased from Santa Cruz Biotechnology. Specific Ambion siRNA silencers for CLEC7A (encoding Dectin-1) (AM16708-249228) and SYK (AM16708-138814) genes were purchased from Thermo Fisher Scientific (Waltham, MA). The immortalized canine airway carcinoma cell line (BACA) was provided by Dr. Timothy Fan (Department of Veterinary Clinical Medicine, College of Veterinary Medicine (CVM), University of Illinois at Urbana-Champaign (UIUC). BACA cells were cultured in the Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution in 5% CO2 at 37°C. After reaching 95% confluency, cells were exposed to either live or heat-killed B. dermatitidis strain SCB-2 (Gilchrist et Stokes; ATCC, Manassas, VA; ATCC 26199) for 24 hours at a multiplicity of infection of 1:1 or 5:1, or to β-glucan at indicated concentrations. B. dermatitidis ATCC 26199 (SCB-2) was maintained as yeast on BD Middlebrook 7H10 agar supplemented with oleic acid–albumin complex (Sigma-Aldrich) slants at 39°C. Histoplasma capsulatum ATCC 26032 (G217B) was grown at 37°C in Ham's F12 medium supplemented with glucose (18.2 g/L), glutamic acid (1 g/L), HEPES (6g/L), and cysteine (8.4 mg/L). The yeasts were heat killed by incubating at 65°C for 30 minutes. The fungal pneumonia cases (Table 1) and lung tissue sections from feline and canine animals were provided by the UIUC CVM Veterinary Diagnostic Laboratory. Healthy control lungs were found to be free of infection on necropsy, and the animals were euthanized of unrelated causes. Animals with fungal pneumonia either died or were euthanized because of infection with B. dermatitidis, H. capsulatum, or C. immitis. Lung tissues were paraffin embedded, divided into sections and stained.Table 1Clinical Cases of Dogs and Cats with Fungal Pneumonia Ana