SPLUNC1 Expression Research Articles

USP7 inhibits the progression of nasopharyngeal carcinoma via promoting SPLUNC1-mediated M1 macrophage polarization through TRIM24

Reprogramming of macrophages toward an M1 phenotype is a novel strategy to induce anticancer immunity. However, the regulatory mechanisms of M1 macrophage polarization and its functional roles in nasopharyngeal carcinoma (NPC) progression need to be further explored. Here we found that SPLUNC1 was highly expressed and responsible for M1 macrophage polarization. JAK/STATs pathway activation was involved in SPLUNC1-mediated M1 macrophage polarization. Importantly, regulation of SPLUNC1 in macrophages affected CM-mediated influence on NPC cell proliferation and migration. Mechanistically, USP7 deubiquitinated and stabilized TRIM24, which promoted SPLUNC1 expression via recruitment of STAT3 in M1 macrophages. Depletion of TRIM24 inhibited M1 macrophage polarization, which facilitated NPC cell growth and migration. However, over-expression of USP7 exhibited the opposite results and counteracted the tumorigenic effect of TRIM24 silencing. Finally, the growth and metastasis of NPC cells in vivo were repressed by USP7-induced M1 macrophage polarization via modulating TRIM24/SPLUNC1 axis. USP7 delayed NPC progression via promoting macrophage polarization toward M1 through regulating TRIM24/SPLUNC1 pathway, providing evidence for the development of effective antitumor immunotherapies for NPC.

Diverse phenotypes and endotypes of fungus balls caused by mixed bacterial colonization in chronic rhinosinusitis.

The pathogenic roles of fungus and bacteria in chronic rhinosinusitis (CRS) remain unclear. Recently, we described the bacterial ball, which is distinct from the fungus ball, as an unusual phenotype of bacterial infection. In this study, we investigated the clinical, histopathologic, and immunologic characteristics of sinonasal microorganic materials, including fungus ball and bacterial ball. In this study, we enrolled 80 CRS patients with sinonasal microorganic materials and 10 control subjects who underwent skull base surgery or endoscopic dacryocystorhinostomy and had no signs or symptoms of nasal inflammation. All specimens were stained with hematoxylin-eosin, Gomori-methenamine-silver, and Gram stain to identify fungal organisms and Gram-positive/negative bacterial colonies. The expression of tumor necrosis factor (TNF)-α; interleukin (IL)-1β; S100A7; S100A8/A9; and short, palate, lung, and nasal epithelial clone 1 (SPLUNC1) were evaluated by enzyme-linked immunosorbent assay using sinus lavage fluid. We histologically classified sinonasal microorganic materials into the following 4 groups: fungus ball (n = 45); bacterial ball (n = 6); mixed ball (formed by a mixture of fungus and bacteria, n = 27); and double ball (formed by separate fungal and bacterial balls, n = 2). Compared with the fungus ball, the mixed ball was more frequently detected in immunocompromised patients (p < 0.0001). In addition, TNF-α expression was significantly higher in fungus and mixed balls than in control, whereas the mixed ball showed higher expression of IL-1β compared with the fungus ball. Moreover, the expression of S100A7 and S100A8/A9 protein in the mixed ball was significantly decreased when compared with the fungus ball, whereas there was no significant difference in SPLUNC1 expression between fungus and mixed balls. Our findings suggest that fungal and bacterial interactions are diverse in CRS. Specifically, the mixed ball is prevalent in CRS with an immunocompromised state and it may decrease epithelial barrier function.

Increased susceptibility to otitis media in a Splunc1-deficient mouse model.

ABSTRACTOtitis media (inflammation of the middle ear) is one of the most common diseases of early childhood. Susceptibility to otitis is influenced by a number of factors, including the actions of innate immune molecules secreted by the epithelia lining the nasopharynx, middle ear and Eustachian tube. The SPLUNC1 (short palate, lung, nasal epithelial clone 1) protein is a highly abundant secretory product of the mammalian nasal, oral and respiratory mucosa that is thought to play a multifunctional role in host defense. In this study we investigated Splunc1 expression in the ear of the mouse, and examined whether this protein contributes to overall host defense in the middle ear and/or Eustachian tube. We found that Splunc1 is highly expressed in both the surface epithelium and in submucosal glands in these regions in wild-type mice. In mice lacking Splunc1, we noted histologically an increased frequency of otitis media, characterized by the accumulation of leukocytes (neutrophils with scattered macrophages), proteinaceous fluid and mucus in the middle ear lumens. Furthermore, many of these mice had extensive remodeling of the middle ear wall, suggesting a chronic course of disease. From these observations, we conclude that loss of Splunc1 predisposes mice to the development of otitis media. The Splunc1−/− mouse model should help investigators to better understand both the biological role of Splunc1 as well as host defense mechanisms in the middle ear.

Splunc Expression in Patients Undergoing Autologous Haematopoietic Stem Cell Trnasplantation

SPLUNC EXPRESSION IN PATIENTS UNDERGOING AUTOLOGOUS HAEMATOPOIETIC STEM CELL TRNASPLANTATION Marcio Ajudarte Lopes, Lara Maria Alencar Ramos, Wilfredo Alejandro Gonzalez Arriagada, Andreia Aparecida Silva, Pablo Agustin Vargas, Ricardo Della Coletta, Lynne Bingle, Oral Diagnosis Department, Piracicaba Dental School, University of Campinas, Brazil; Oral and Maxillofacial Pathology, School of Clinical Dentistry, University of Sheffield, Sheffield, UK Objective: The aim of this study was to analyze flow rate and SPLUNC2 and SPLUNC1 expression in patients who underwent high-dose chemotherapy (HDT) followed by autologous hematopoietic stem cell transplantation (autologous HSCT). Study Design: A sample of autologous HSCT patients was analyzed. Unstimulated saliva was collected before high dose therapy (HDT), on D+7, and on D+21 and SPLUNC expression was analyzed by western blotting. Results: Fifteen patients were included in this study, (mean age 46 14), 9 men and 6 women. Densitometry of protein bands showed higher level of SPLUNC2 after transplant on D+7 compared with before HDT and reduction on D+21. SPLUNC1 also increased after transplant on D+7 compared with pre-HDT and reduction on D+21. In addition, salivary flow rate decreased after autologous HSCT. Conclusions: The present study shows that patients who underwent HDT for autologous HSCT may have changes in SPLUNC2 and SPLUNC1 expression and in salivary flow rate.

Salivary SPLUNC2A and SPLUNC1 Expression is Modified by Head and Neck Cancer Radiotherapy and Can be Associated with Collateral Effects

Salivary SPLUNC2A and SPLUNC1 Expression is Modified by Head and Neck Cancer Radiotherapy and Can be Associated with Collateral Effects

SPLUNC1 is associated with nasopharyngeal carcinoma prognosis and plays an important role in all-trans-retinoic acid-induced growth inhibition and differentiation in nasopharyngeal cancer cells.

Human SPLUNC1 can suppress nasopharyngeal carcinoma (NPC) tumor formation; however, the correlation between SPLUNC1expression and NPC patient prognosis has not been reported. In the present study, we used a large-scale sample of 1015 tissue cores to detect SPLUNC1 expression and its association with patient prognosis. SPLUNC1 expression was reduced in NPC samples compared to nontumor nasopharyngeal epithelium tissues. Positive expression of SPLUNC1 in NPC predicted a better prognosis (disease-free survival, P = 0.034; overall survival, P = 0.048). Cox's proportional hazards model revealed that SPLUNC1 could be a significant prognostic factor affecting disease-free survival (P = 0.027). A cDNA micro-array analyzed by significant analysis of micro-array (SAM) and ingenuity pathway analysis (IPA) revealed that an indirect interaction existed between SPLUNC1 and retinoic acid (RA) in the cancer regulatory network. To further investigate the molecular mechanisms involved, we utilized several bioinformatics tools and identified 12 retinoid X receptors heterodimer binding sites in the promoter region of the SPLUNC1 gene. The transcriptional activity of the SPLUNC1 promoter was up-regulated significantly by all-trans-retinoic acid (ATRA). SPLUNC1 and retinoic acid receptor expression were induced significantly by ATRA, and removal of ATRA led to a progressive loss of SPLUNC1 and retinoic acid receptor expression. ATRA inhibited proliferation and induced the differentiation of NPC cells. Interestingly, over-expression of SPLUNC1 sensitized NPC cells to ATRA, whereas knockdown of SPLUNC1 in HNE1 cells increased cell viability. Under SPLUNC1 knockdown conditions, differentiation was reversed by ATRA treatment. We concluded that SPLUNC1 could potentially predict prognosis for NPC patients and play an important role in ATRA-induced growth inhibition and differentiation in NPC cells.

Electronic cigarette liquid increases inflammation and virus infection in primary human airway epithelial cells.

Background/ObjectiveThe use of electronic cigarettes (e-cigarettes) is rapidly increasing in the United States, especially among young people since e-cigarettes have been perceived as a safer alternative to conventional tobacco cigarettes. However, the scientific evidence regarding the human health effects of e-cigarettes on the lung is extremely limited. The major goal of our current study is to determine if e-cigarette use alters human young subject airway epithelial functions such as inflammatory response and innate immune defense against respiratory viral (i.e., human rhinovirus, HRV) infection.Methodology/Main ResultsWe examined the effects of e-cigarette liquid (e-liquid) on pro-inflammatory cytokine (e.g., IL-6) production, HRV infection and host defense molecules (e.g., short palate, lung, and nasal epithelium clone 1, SPLUNC1) in primary human airway epithelial cells from young healthy non-smokers. Additionally, we examined the role of SPLUNC1 in lung defense against HRV infection using a SPLUNC1 knockout mouse model. We found that nicotine-free e-liquid promoted IL-6 production and HRV infection. Addition of nicotine into e-liquid further amplified the effects of nicotine-free e-liquid. Moreover, SPLUNC1 deficiency in mice significantly increased lung HRV loads. E-liquid inhibited SPLUNC1 expression in primary human airway epithelial cells. These findings strongly suggest the deleterious health effects of e-cigarettes in the airways of young people. Our data will guide future studies to evaluate the impact of e-cigarettes on lung health in human populations, and help inform the public about potential health risks of e-cigarettes.

The effects of physical exercise and smoking habits on the expression of SPLUNC1 in nasal lavage fluids from allergic rhinitis subjects

ObjectivePalate lung nasal epithelial clone (PLUNC) is a family of proteins, which are proposed to participate in the innate immune defense against infections in the upper aero-digestive tract. The aim of this study was to investigate the expression of SPLUNC1 in allergic rhinitis subjects with considerations taken to the mucosal function and smoking habits. MethodsThe participants, recruited from a cohort followed from infancy, were re-examined at the age of 18 years regarding allergy development. Based on medical histories and skin prick tests the participants were classified into groups with persistent allergic rhinitis (n=18), intermittent allergic rhinitis (n=8) and healthy controls (n=13). Seven subjects (3, 2 and 2 in each group, respectively) reported smoking habits. The SPLUNC1 levels in nasal lavage fluids were analyzed by Western blot. Changes in the volume of the proper nasal cavity before and after physical exercise (Vol2increase) were analyzed by acoustic rhinometry. ResultsCompared to the control group the SPLUNC1 level was significantly lower in the persistent allergy group (3.8±3.4 OD vs. 1.3±1.5 OD; p=0.02), but not in the intermittent allergy group without current exposure to allergens (3.6±4.7 OD). No differences were found in Vol2increase between any of the allergy groups and controls. In smokers Vol2increase was significantly reduced (p<0.01) and the SPLUNC1 levels were lower compared to non-smokers. A significant correlation was found between SPLUNC1 and Vol2increase (p<0.01; r=0.53) in non-smokers. ConclusionsCurrent allergen exposure has an impact on SPLUNC1 expression in nasal lavage fluid, why allergy ought to be considered in study populations where analyses of SPLUNC1 levels are included in the reports. The normal nasal decongestion after exercise was not affected by allergy in contrast to smoking habits. The correlation between SPLUNC1 levels and Vol2increase in non-smokers may indicate involvement of SPLUNC1in the regulation of the normal function of the nasal mucosa. Complementary studies are needed to confirm the smoke-related reduction of SPLUNC1 expression and to analyze the possible participation of SPLUNC1 in the nasal mucosa regulation.

Increased Susceptibility to Pulmonary Pseudomonas Infection in Splunc1 Knockout Mice

The airway epithelium is the first line of host defense against pathogens. The short palate, lung, and nasal epithelium clone (SPLUNC)1 protein is secreted in respiratory tracts and is a member of the bacterial/permeability increasing (BPI) fold-containing protein family, which shares structural similarities with BPI-like proteins. On the basis of its homology with BPIs and restricted expression of SPLUNC1 in serous cells of submucosal glands and surface epithelial cells of the upper respiratory tract, SPLUNC1 is thought to possess antimicrobial activity in host defense. SPLUNC1 is also reported to have surfactant properties, which may contribute to anti-biofilm defenses. The objective of this study was to determine the in vivo functions of SPLUNC1 following Pseudomonas aeruginosa infection and to elucidate the underlying mechanism by using a knockout (KO) mouse model with a genetic ablation of Splunc1. Splunc1 KO mice showed accelerated mortality and increased susceptibility to P. aeruginosa infection with significantly decreased survival rates, increased bacterial burdens, exaggerated tissue injuries, and elevated proinflammatory cytokine levels as compared with those of their wild-type littermates. Increased neutrophil infiltration in Splunc1 KO mice was accompanied by elevated chemokine levels, including Cxcl1, Cxcl2, and Ccl20. Furthermore, the expression of several epithelial secretory proteins and antimicrobial molecules was considerably suppressed in the lungs of Splunc1 KO mice. The deficiency of Splunc1 in mouse airway epithelium also results in increased biofilm formation of P. aeruginosa. Taken together, our results support that the ablation of Splunc1 in mouse airways affects the mucociliary clearance, resulting in decreased innate immune response during Pseudomonas-induced respiratory infection.

Regional differences in the expression of innate host defense molecules in sinonasal mucosa

Nasal mucosal epithelial cells form the first line of defense by maintaining a barrier to restrict potentially harmful airborne substances and pathogens. To aid in host defense, specialized epithelial cells in submucosal glands as well as on the mucosal surface secrete mucous that helps to immobilize pathogens and other harmful substances. Beneath this mucous blanket resides an aqueous surface liquid layer, which allows proper ciliary function to clear mucous and entrapped pathogens. In concert with maintenance of a physical barrier, airway epithelium secretes dozens of antimicrobial peptides and proteins that become incorporated into the mucous and aqueous lining fluids1, 2. It has become clear that proper functioning of the epithelium is essential for suppressing the growth of pathogenic organisms and promoting healthy upper airway physiology. Dysfunction in innate immune expression of host defense molecules has been linked to many airway diseases3-8. Surprisingly, the regional distribution of important epithelial derived antimicrobial proteins secreted into upper airways has not been studied in detail. In this study, we analyzed the expression of select antimicrobial proteins that are known to be secreted into the lining fluids of the upper airways in two different anatomical sites of the sinonasal mucosa in healthy human subjects, namely the inferior region (inferior turbinate - IT), and the superior region (uncinate tissue - UT). We chose IT, as it is the proximal point of contact of inhaled air containing particulate matter and potential pathogens. Moreover, studies of CRS and other diseases of the upper airways routinely use IT tissue as a control tissue to make comparisons with nasal polyps. UT was chosen for its important role in the drainage pathways of maxillary, frontal and anterior ethmoid sinuses. We chose to study the expression of S100A7 (S100 family), hBD2 (β-Defensin family), SPLUNC1 (PLUNC family), and lactoferrin, as they represent broad families of antimicrobial proteins, which are either constitutively present or induced by inflammation triggered by pathogens and collectively have antimicrobial effects against a variety of pathogens (bacteria, fungi and viruses). Sinonasal tissue samples from IT and UT were collected from subjects undergoing surgery to correct non-inflammatory conditions, including facial anatomical defects (deformity, trauma etc.), to improve airflow and during the course of skull base surgery to remove tumors. The subjects were not diagnosed with any upper or lower airway diseases at the time of sample collection. Detailed patient characteristics are provided in table EI. The Investigational Review Board of Northwestern University approved all methods for the present study, and all patients provided informed consent. At the time of surgery, tissues and nasal epithelial scraping cells were collected and stored for further analysis. Tissue samples and epithelial cells were analyzed for mRNA expression by real time PCR. Protein expression and localization were analyzed using ELISA and immunohistochemistry, respectively. AB/PAS staining was performed to characterize the glandular differences between IT and UT. Details of the methods used can be found in the online repository and our other publications5, 7.

WS20.4 The rs1078761 polymorphism is associated with reduced SPLUNC1 expression and increased pulmonary disease severity in cystic fibrosis patients

WS20.4 The rs1078761 polymorphism is associated with reduced SPLUNC1 expression and increased pulmonary disease severity in cystic fibrosis patients

PP010: Analysis of SPLUNC1 expression in saliva of patients under chemotherapy and radiotherapy for head and neck cancer

Purpose SPLUNC1 is a protein that participates in the host innate immunity, mainly expressed in the respiratory tract epithelium, secretions, salivary glands and saliva. Its expression was also described in infections, inflammatory disorders, and malignancies. Patients who underwent chemotherapy (CT) or radiotherapy (RT) for head and neck tumors may suffer important oral side effects, such as mucositis, candidosis, taste alterations and hypossalivation, which can interfere with SPLUNC1 expression. Therefore, the purpose of this study was to analyze the saliva of patients who underwent CT and/or RT and describe the association of SPLUNC1 expression and collateral effects of the therapy. Material and methods A prospective randomized study was performed with two groups, chemotherapy patients ( n = 10) and head and neck radiotherapy patients ( n = 10). Unstimulated saliva for 5 min was collected before, during, and after the treatment. Clinical data and collateral effects were obtained from the patients’ charts. SPLUNC1 expression was analyzed by western blotting, and densitometry was performed. The results were statistically analyzed (ANOVA, Tukey’s, p Results Twenty patients were included in this study, (mean age of 51 years). It was observed that 60% of the patients who received RT presented hypossalivation after treatment ( p = 0.001). Higher level of SPLUNC1 occurred in RT patients who had mucositis and candidosis ( p = 0.037), in all the three collected samples. On the other hand, SPLUNC1 proteins decreased in patients submitted to chemotherapy without correlation with any side effect. Conclusions The present study describes recent findings about expression of SPLUNC1 in cancer patients who underwent chemotherapy and head and neck radiotherapy before and after the treatment. Despite the small sample, this study suggests that changes in the SPLUNC1 expression could be associated with oral side effects during radiotherapy and chemotherapy.

Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis

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. 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. 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. 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.

Expression of CCSP and SPLUNC1, Putative Anti-Inflammatory Proteins, Following Murine Respiratory Viral Infection In Vivo

Expression of CCSP and SPLUNC1, Putative Anti-Inflammatory Proteins, Following Murine Respiratory Viral Infection In Vivo

Identification of a functional variant in SPLUNC1 associated with nasopharyngeal carcinoma susceptibility among Malaysian Chinese

Nasopharyngeal carcinoma (NPC) is a multifactorial and polygenic disease with high incidence in Asian countries. Epstein-Barr virus infection, environmental and genetic factors are believed to be involved in the tumorigenesis of NPC. The association of single nucleotide polymorphisms (SNPs) in LPLUNC1 and SPLUNC1 genes with NPC was investigated by performing a two-stage case control association study in a Malaysian Chinese population. The initial screening consisted of 81 NPC patients and 147 healthy controls while the replication study consisted of 366 NPC patients and 340 healthy controls. The combined analysis showed that a SNP (rs2752903) of SPLUNC1 was significantly associated with the risk of NPC (combined P = 0.00032, odds ratio = 1.62, 95% confidence interval = 1.25-2.11). In the subsequent dense fine mapping of SPLUNC1 locus, 36 SNPs in strong linkage disequilibrium with rs2752903 (r(2) ≥ 0.85) were associated with NPC susceptibility. Screening of these variants by electrophoretic mobility shift and luciferase reporter assays showed that rs1407019 located in intron 3 (r(2) = 0.994 with rs2752903) caused allelic difference in the binding of specificity protein 1 (Sp1) transcription factor and affected luciferase activity. This SNP may consequently alter the expression of SPLUNC1 in the epithelial cells. In summary, our study suggested that rs1407019 in intronic enhancer of SPLUNC1 is associated with NPC susceptibility in which its A allele confers an increased risk of NPC in the Malaysian Chinese population.

Alterations in SPLUNC1 expression in rodent models of respiratory virus infection

Alterations in SPLUNC1 expression in rodent models of respiratory virus infection

SPLUNC1 expression reduces surface levels of the epithelial sodium channel (ENaC) in Xenopus laevis oocytes

Throughout the body, the epithelial Na+ channel (ENaC) plays a critical role in salt and liquid homeostasis. In cystic fibrosis airways, for instance, improper regulation of ENaC results in hyperabsorption of sodium that causes dehydration of airway surface liquid. This dysregulation then contributes to mucus stasis and chronic lung infections. ENaC is known to undergo proteolytic cleavage, which is required for its ability to conduct Na+ ions. We have previously shown that the short, palate lung and nasal epithelial clone (SPLUNC1) binds to and inhibits ENaC in both airway epithelia and in Xenopus laevis oocytes. In this study, we found that SPLUNC1 was more potent at inhibiting ENaC than either SPLUNC2 or long PLUNC1 (LPLUNC1), two other PLUNC family proteins that are also expressed in airway epithelia. Furthermore, we were able to shed light on the potential mechanism of SPLUNC1's inhibition of ENaC. While SPLUNC1 did not inhibit proteolytic activity of trypsin, it significantly reduced ENaC currents by reducing the number of ENaCs in the plasma membrane. A better understanding of ENaC's regulation by endogenous inhibitors may aid in the development of novel therapies designed to inhibit hyperactive ENaC in cystic fibrosis epithelia.

SPLUNC1 expression reduces surface levels of the epithelial sodium channel (ENaC) in Xenopus laevis oocytes

SPLUNC1 expression reduces surface levels of the epithelial sodium channel (ENaC) in Xenopus laevis oocytes

Ontogeny of mRNA expression of pulmonary innate immune factors

Prematurely born infants are at an increased risk of developing respiratory infections due in part to decreased function of their innate immune system. It was our goal to examine the ontogeny of innate immune gene expression by respiratory epithelia in our ovine model. Targets of interest are surfactant proteins A and D (SP‐A and SP‐D), sheep beta defensin 1 (SBD‐1), dual oxidases 1 and 2 (Duox1 and Duox2), lactoperoxidase (LPO), SPLUNC1, LPLUNC2, Toll‐like receptors 3, 7, and 8 (TLR 3, 7, and 8). Lung from sheep was collected at 115 and 130 days gestation, full term, 15 days post partum, and adulthood. Levels of mRNA for the target innate immune genes were measured by real‐time RT‐qPCR. LPO, SPLUNC1, LPLUNC2, TLR‐8, SBD‐1, and Duox1 expression increased progressively with age. Surfactant proteins A and D, previously reported to be decreased in preterm lambs, did not follow a steady trend, although SP‐A expression was lower at 130 days gestation than at full term. Duox2 levels were lower in gestational and perinatal lambs than in adults, but did not show a linear increase with age. These results are significant in that they may indicate that reduced expression in fetal lung may underlie susceptibility of the lung to infection with preterm birth. Conversely, upregulation of these genes may have therapeutic potential.