The multifunctional host defense peptide SPLUNC1 is critical for homeostasis of the mammalian upper airway

Abstract

Otitis media (OM) is a highly prevalent pediatric disease caused by normal flora of the nasopharynx that ascend the Eustachian tube and enter the middle ear. As OM is a disease of opportunity, it is critical to gain an increased understanding of immune system components that are operational in the upper airway and aid in prevention of this disease. SPLUNC1 is an antimicrobial host defense peptide that is hypothesized to contribute to the health of the airway both through bactericidal and non-bactericidal mechanisms. We used small interfering RNA (siRNA) technology to knock down expression of the chinchilla ortholog of human SPLUNC1 (cSPLUNC1) to begin to determine the role that this protein played in prevention of OM. We showed that knock down of cSPLUNC1 expression did not impact survival of nontypeable Haemophilus influenzae, a predominant causative agent of OM, in the chinchilla middle ear under the conditions tested. In contrast, expression of cSPLUNC1 was essential for maintenance of middle ear pressure and efficient mucociliary clearance, key defense mechanisms of the tubotympanum. Collectively, our data have provided the first in vivo evidence that cSPLUNC1 functions to maintain homeostasis of the upper airway and, thereby, is critical for protection of the middle ear.

Figure 1. cSPLUNC1 mRNA and protein expression in the chinchilla upper airway.

(A) RT-PCR analysis of (top panel) cSPLUNC1 or (bottom panel) β-actin transcripts in multiple chinchilla tissue homogenates. β-actin mRNA was amplified to confirm that equal amounts of template RNA was used in the amplification process. Amplicons generated with reverse transcriptase (+) or without reverse transcriptase (−) were separated in an ethidium bromide-stained agarose gel. (B) We used anti-human SPLUNC1 in Western blot to detect native cSPLUNC1 in selected mucosal homogenates. Recombinant cSPLUNC1 [(r)cSPLUNC1] was also detected to provide evidence that the antisera used in this analysis recognized the chinchilla ortholog of human SPLUNC1 and recombinant human SPLUNC1 [(r)hSPLUNC1] served as the positive control in this analysis. (C) Proteins in nasopharyngeal lavage fluid were separated by SDS-PAGE and Western blot was used to detect secreted cSPLUNC1 in the upper airway. cSPLUNC1 mRNA and protein was detected in every URT tissue evaluated.

Figure 2. Detection of siRNA in the chinchilla upper airway.

A negative control siRNA labeled with Alexafluor-647 was administered intranasally and transbullarly to chinchillas and fluorescence was detected (A) immediately, (B, C, and D) three hours, or (E) five days after delivery of siRNA. The images in (B and C) are of a sagittal section of a chinchilla head with tissues that express cSPLUNC1 identified by arrows. The nasoturbinates in (C) are covered by black non-fluorescent paper to block the strong fluorescence from this tissue and to allow detection of the weaker fluorescence from siRNA in the ethmoid turbinates and nasopharynx. (D) Nasoturbinate mucosae was embedded, sectioned, and stained with phalloidin conjugated to FITC to detect actin (green) and DAPI to stain DNA (blue). The top panel shows the fluorescently-labeled siRNA (red) alone and the bottom panel shows labeling for siRNA, actin, and DAPI. (E, right panel) Ex vivo analysis of the presence of siRNA in the tubotympanum. These data demonstrated that siRNA entered epithelial cells of the upper airway and showed our ability to detect siRNA in the chinchilla nasal cavity, Eustachian tube, and middle ear for at least five days after delivery of siRNA.

Figure 3. Knock down of cSPLUNC1 expression in vivo.

(A) NL fluids were collected from chinchillas prior to administration of siRNA (d = 0) or after delivery of 10 nmoles of an siRNA. Two unique siRNAs that were designed based on the cDNA sequence of cSPLUNC1 and an siRNA that did not have complementary sequence to cSPLUNC1 were intranasally delivered to chinchillas, and NL fluids were collected from animals one and five days later. cSPLUNC1 protein present in NL fluids was detected by Western blot using rabbit anti-native cSPLUNC1 and knock down in expression was determined by comparison of band intensities for each panel before and after delivery of siRNA. (B) The siRNA molecules utilized in (A) were administered intranasally to chinchillas (n = 3 per cohort) and 24 hours later animals were sacrificed, nasoturbinate mucosae was collected, and total RNA was isolated. RT-PCR with primers designed to amplify (top panel) cSPLUNC1 or (middle and bottom panels) the control β-actin was used to determine relative mRNA expression for the targets among the cohorts. Amplification was also done in the absence of reverse transcriptase to demonstrate the absence of contaminating DNA as shown in the bottom panel. (C) Densitometric analysis of the intensity of RT-PCR amplicons shown in (B). Administration of 10 nmoles of cSPLUNC1 siRNA #1 resulted in a significant reduction (asterisk) in cSPLUNC1 expression, compared to the negative control siRNA.

Figure 4. Purification of native cSPLUNC1 and its ability to kill NTHI.

Secreted proteins obtained from washing the apical surface of CNPEs (Fig. S2) with PBS were separated by SDS-PAGE and (A) silver stained or (B) used in Western blot to show native cSPLUNC1. Additionally, proteins collected from the surface of CNPEs were separated by SDS-PAGE, and cSPLUNC1 was gel extracted, electroeluted, and refolded to obtain native protein. (C) Purified cSPLUNC1 was detected in a silver stained SDS-PAGE gel and (D) shown to exhibit secondary structure as determined by circular dichroism analysis. (E) NTHI 86-028NP was incubated with increasing concentrations of native cSPLUNC1, LL-37, or lysozyme for 1 hour, and the number of surviving colony forming units were determined. Native cSPLUNC1 demonstrated bactericidal activity against NTHI in vitro which suggested that cSPLUNC1 may also impact the ability of NTHI to survive in vivo.

Figure 5. Effect of cSPLUNC1 knock down on the development of OM induced by NTHI.

Chinchillas (3 per cohort) were administered either a control or cSPLUNC1 specific siRNA and challenged one day later with NTHI. The load of NTHI in middle ear effusions was subsequently determined two and four days after bacterial challenge. Knock down in cSPLUNC1 did not significantly impact survival of NTHI in the chinchilla middle ear under the conditions tested wherein a rigorous challenge dose was used.

Figure 6. Impact of cSPLUNC1 knock down on the tubotympanum.

Chinchillas were administered either a control or cSPLUNC1-specific siRNA and (A) tympanometry was used to determine middle ear pressure and tympanic membrane compliance and (B) computed tomography imaging was used to visualize the right middle ear cavity of chinchillas 24 hours after delivery of siRNA (before challenge with NTHI). The tympanic membrane in (B) was pseudo-colored red to highlight the retraction of the tympanic membrane that occurred in animals that received cSPLUNC1 siRNA. Other structural differences that are observed between the left and right panels (ie. morphology of the cochlea) are due to slight differences in the positioning of animals in the scanner and do not represent pathological changes in the ears of animals when expression of cSPLUNC1 was diminished. Images shown in (A) and (B) are representative of results obtained from the respective cohorts. Knock down of cSPLUNC1 resulted in a strong middle ear under pressure as evidenced by marked retraction of the tympanic membrane. EAC – external ear canal, ME- middle ear cavity, C- cochlea.

Figure 7. Effect of cSPLUNC1 knock down on the histopathology of the ET recovered from animals with NTHI-induced OM.

Chinchillas (n = 2 per cohort) were administered either (A) a control or (B) cSPLUNC1-specific siRNA and challenged one day later with NTHI. After four days, animals were sacrificed and Eustachian tubes were embedded and stained with hematoxylin and eosin. Asterisks in (B) indicate goblet cells. Knock down in cSPLUNC1 expression in chinchillas with OM induced by NTHI resulted in a pronounced influx of inflammatory cells, accumulation of cellular debris and mucus, and goblet cell hyperplasia in the ET.

Figure 8. Surfactant activity of native cSPLUNC1.

The relative ability of a droplet of water, lysozyme, native cSPLUNC1 or SDS to exhibit surfactant activity was determined via (A, B, and C) contact angle or (D) pendant drop tensiometry analysis. SDS was used as a positive control for surfactant activity whereas water and lysozyme served as the negative controls. (A, B, and C) Droplets of the four solutions were incubated on a hydrophobic surface and images from a (A) top or (C) side view were collected. (B) The diameter of each droplet was measured in millimeters (n = 4) and compared, as a percentage, to the water control which was set to a value of zero. (D) Surface tension of the four solutions was determined (n = 5) and the mean ± standard deviation presented. Asterisks denote a statistically significant difference (p<0.05) in values between samples and the water alone control. Similar to SDS, native cSPLUNC1 reduced surface tension of water and suggested that this AP acted as a surfactant in vivo.

Figure 9. Role of cSPLUNC1 in ET function.

Chinchillas (4 ears per cohort) were administered either saline, isoproterenol, cSPLUNC1 siRNA, or a negative control siRNA followed by delivery of a very small volume of dye into the middle ear cavity 24 hours later. The average transport time ± standard deviation (in seconds) required for dye to be transported from the middle ear to the nasopharyngeal orifice of the ET was determined for each cohort. Asterisks denote a statistically significant (p≤0.05) difference in dye transport time between the cohort that received saline alone and the cohorts that received isoproterenol or the cSPLUNC1 siRNA. Whereas the β-adrenergic mucociliary stimulator isoproterenol increased transport time, knock down in cSPLUNC1 expression conversely prolonged dye transport time likely as a result of reduced surfactant activity which diminished ET mucociliary clearance.