Altered Mitochondrial Functions and Morphologies in Epithelial Cells Are Associated With Pathogenesis of Chronic Rhinosinusitis With Nasal Polyps

Abstract

Purpose: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a complex inflammatory disease of the nasal and paranasal sinus mucosa. The disease is associated with mitochondrial dysfunction, structural changes in the mitochondria, and reactive oxygen species (ROS) generation. This study investigated whether there are functional and morphological changes in the mitochondria in the epithelial cells of nasal polyps (NPs) and Staphylococcus aureus enterotoxin B (SEB)-stimulated nasal epithelial cells.

Methods: In all, 30 patients with CRSwNP and 15 healthy subjects were enrolled. Mitochondrial ROS (mtROS) and changes in mitochondrial functions and structures were investigated in the uncinate tissue (UT) of healthy controls, the UT or NPs of CRSwNP patients, and human nasal epithelial cells with or without SEB stimulation.

Results: Oxidative phosphorylation complexes showed various responses following SEB stimulation in the nasal epithelial cells, and their expressions were significantly higher in the NPs of patients with CRSwNP than in the UT of controls. Generation of mtROS was increased following SEB exposure in nasal epithelial cells and was reduced by pretreatment with MitoTEMPO, which is used as an mtROS scavenger. In the tissues, mtROS was significantly increased in the NPs of CRSwNP patients compared to the UT of controls or CRSwNP patients. The expressions of fusion- and fission-related molecules were also significantly higher in SEB-exposed nasal epithelial cells than in non-exposed cells. In tissues, the expression of fission (fission mediator protein 1)- and fusion (membrane and mitofusin-1, and optic atrophy protein 1)-related molecules was significantly higher in the NPs of CRSwNP patients than in UT of controls or CRSwNP patients. Transmission electron microscopy revealed elongated mitochondria in SEB-exposed nasal epithelial cells and epithelial cells of NPs.

Conclusions: Production of mtROS, disrupted mitochondrial function, and structural changes in nasal epithelial cells might be involved in the pathogenesis of CRSwNP.

Keywords: Rhinitis; Staphylococcus; enterotoxin; epithelial cells; mitochondria; nasal polyps; reactive oxygen species.

Fig. 1. Expression of oxidative phosphorylation complex enzymes and OCRs. (A) Oxidative phosphorylation complex I–V expression levels in RPMI 2650 cells exposed to different doses of SEB for 48 hours. (B) Oxidative phosphorylation complex I–V expression levels in NP tissue (control, n = 4; UT, n = 8; NP, n = 8). (C) OCRs in RPMI 2650 cells exposed to SEB (5 μg, 48 hours) and SEB (5 μg, 48 hours) with MitoTEMPO treatment (10 μmol/L).

OCR, oxygen consumption rate; SEB, Staphylococcus aureus enterotoxin B; CCCP, carbonyl cyanide m-chlorophenyl hydrazine; UT, uncinate tissue; NP, nasal polyp. ^*P < 0.05; ^†P < 0.01; ^‡P < 0.005; ^§P < 0.001.

Fig. 2. The mtROS and Mn-SOD expression in nasal epithelial cells and NP tissue. Confocal images showing mtROS expression using MitoSOX after exposure to 0 and 5 µg of SEB (48 hours) on human nasal epithelial cells (RPMI 2650) (A) and pHNECs (B) with the presence and absence of MitoTEMPO (10 μmol/L). Mn-SOD expression levels in RPMI 2650 cells (C) and pHNECs (D) following SEB exposure with MitoTEMPO treatment. (E) Mn-SOD expression in NP tissues (n = 6). (F) Immunofluorescence image (×400) of Mn-SOD in NP tissue (control, n = 3; NE-NP, n = 4; E-NP, n = 4).

mtROS, mitochondrial ROS; SEB, Staphylococcus aureus enterotoxin B; Mn-SOD, manganese-dependent superoxide dismutase; NP, nasal polyp; pHNEC, primary human nasal epithelial cell; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; UT, uncinate tissue; DAPI, 4′,6-diamidino-2-phenylindole; NE-NP, non-eosinophilic nasal polyp; E-NP, eosinophilic nasal polyp. ^*P < 0.05, ^†P < 0.01.

Fig. 3. The membrane potential of mitochondria in nasal epithelial cells. Confocal microscope image showing the membrane potential status of mitochondria using MitoTracker Red in RPMI 2650 cells exposed to 0 and 5 µg of SEB for 48 hours with or without MitoTEMPO treatment (10 μmol/L).

SEB, Staphylococcus aureus enterotoxin B; DAPI, 4′,6-diamidino-2-phenylindole. ^*P < 0.05, ^†P < 0.01.

Fig. 4. The mRNA expression of fission and fusion markers. (A) The effects of SEB on mRNA expression of fission- and fusion-related genes in RPMI 2650 cells exposed to different doses of SEB. (B) The mRNA expression of fission- and fusion-related genes in NP tissue (control, n = 4; UT, n = 4; NP, n = 4).

SEB, Staphylococcus aureus enterotoxin B; mRNA, messenger RNA; NP, nasal polyp; UT, uncinate tissue; Drp1, dynamin-related protein 1; Fis1, fission mediator protein 1; Mfn1, membrane and mitofusin-1; OPA1, optic atrophy protein 1. ^*P < 0.05, ^†P < 0.01, ^‡P < 0.005.

Fig. 5. Transmission electron micrograph of mitochondria. The mitochondria exhibited various sizes and morphologies in the epithelial cells of the control tissue (A, B). However, in the epithelial cells of NP, increased numbers of fragmented mitochondria with vacuoles (arrow in C) and mitochondria with unified elongated morphology (dotted line in D) were observed. In SEB-exposed human nasal epithelial cells, the number of elongated mitochondria (black arrow in F), which are indicative of a hyper-fusion state, was also increased compared with control (E, F).

SEB, Staphylococcus aureus enterotoxin B; UT, uncinate tissue; NP, nasal polyp.

Fig. 6. PINK1 expression. PINK1 mRNA expression in human nasal epithelial cells (RPMI 2650) (A) and primary human nasal epithelial cells. (B) Exposure to SEB (5 μg, 48 hours) with or without MitoTEMPO treatment (10 μmol/L). (C) PINK1 mRNA expression in NP tissue (control, n = 4; UT, n = 5; NP, n = 5).

SEB, Staphylococcus aureus enterotoxin B; UT, uncinate tissue; NP, nasal polyp; PINK1, PTEN induced kinase 1; mRNA, messenger RNA. ^*P < 0.05; ^†P < 0.01.