Targeting mitochondria-derived reactive oxygen species to reduce epithelial barrier dysfunction and colitis

Abstract

Epithelial permeability is often increased in inflammatory bowel diseases. We hypothesized that perturbed mitochondrial function would cause barrier dysfunction and hence epithelial mitochondria could be targeted to treat intestinal inflammation. Mitochondrial dysfunction was induced in human colon-derived epithelial cell lines or colonic biopsy specimens using dinitrophenol, and barrier function was assessed by transepithelial flux of Escherichia coli with or without mitochondria-targeted antioxidant (MTA) cotreatment. The impact of mitochondria-targeted antioxidants on gut permeability and dextran sodium sulfate (DSS)-induced colitis in mice was tested. Mitochondrial superoxide evoked by dinitrophenol elicited significant internalization and translocation of E. coli across epithelia and control colonic biopsy specimens, which was more striking in Crohn's disease biopsy specimens; the mitochondria-targeted antioxidant, MitoTEMPO, inhibited these barrier defects. Increased gut permeability and reduced epithelial mitochondrial voltage-dependent anion channel expression were observed 3 days after DSS. These changes and the severity of DSS-colitis were reduced by MitoTEMPO treatment. In vitro DSS-stimulated IL-8 production by epithelia was reduced by MitoTEMPO. Metabolic stress evokes significant penetration of commensal bacteria across the epithelium, which is mediated by mitochondria-derived superoxide acting as a signaling, not a cytotoxic, molecule. MitoTEMPO inhibited this barrier dysfunction and suppressed colitis in DSS-colitis, likely via enhancing barrier function and inhibiting proinflammatory cytokine production. These novel findings support consideration of MTAs in the maintenance of epithelial barrier function and the management of inflammatory bowel diseases.

Figure 1

Schematic of the experimental protocols used to assess the impact of systemic MitoTEMPO on DSS-induced colitis in BALB/c mice. MitoTEMPO was used in a prophylactic regimen in which mice underwent necropsy 3 (I) or 5 (II) days after DSS or after 5 days of DSS and then 3 days of normal water (III). When used in a treatment regimen, MitoTEMPO was delivered after removal of the DSS water and animals underwent necropsy on day 8 (IV).

Figure 2

Perturbed epithelial mitochondrial activity reduces barrier function. Confluent filter-grown T84 epithelial cell monolayers display reduced TER 24 hours after exposure to E. coli (Ec; strain HB101, 10⁶ CFU inoculum) that is enhanced by 0.1 mmol/L DNP, 1 μmol/L rotenone (Rot), or 1 μmol/L oligomycin A (OmA) cotreatment (n = 3 experiments, nine epithelial preparations) (A, top panel). Epithelial ATP levels 16 hours after treatment (n = 9 to 12 monolayers from two experiments) (A, bottom panel). E. coli translocation (B) and internalization (C) across filter-grown T84 cell monolayers are significantly increased by DNP, Rot, or OmA cotreatment, as is E. coli internalization into plastic-grown T84 epithelia (n = 3 to 4 experiments, 8 to 13 epithelial preparations) (D). E: After killing extracellular bacteria, significant numbers of E. coli are recovered in the culture medium from DNP-treated epithelial monolayers, which is reduced by treatment with the mitochondria-targeted antioxidants, 10 μmol/L MitoQ (mQ) or 10 μmol/L MitoTEMPO (mT) (n = 4 epithelial monolayers from one representative experiment of four experiments). F: Application of 0.5 mmol/L H₂O₂ directly to plastic-grown T84 cell monolayers does not evoke increased internalization of E. coli (n = 3 experiments, 9 monolayers/condition). Data are given as means ± SEM. ^∗P < 0.05 versus Ec, ^†P < 0.05 versus control (con), and ^‡P < 0.05 versus Ec + DNP.

Figure 3

Targeting mitochondria-derived superoxide reduces bacterial transcytosis across model epithelia. A: Representative epifluorescence images showing mitochondria (Mitotracker) and that mitochondria-derived superoxide (MitoSox) is a rapid (30 minutes) and sustained (16 hours) response to DNP (0.1 mmol/L); and this response is greatest after treatment with DNP and E. coli (Ec; inoculum at 10⁶ CFU). B: The decrease in TER elicited by DNP + E. coli is partially corrected by cotreatment with the mitochondria-targeted antioxidants, 10 μmol/L MitoQ (mQ), or 10 μmol/L MitoTEMPO (mT) (starting TER range = 984 to 1974 Ω cm²). MitoTEMPO completely blocks the increased bacterial internalization and translocation of E. coli across filter-grown T84 monolayers (n = 12 epithelia from four experiments) (C) and internalization into plastic-grown epithelia induced by DNP (n = 9 epithelia from three experiments) (D). Barrier studies were conducted at 16 hours after treatment; effect of 5 mmol/L N-acetylcysteine (NAC) as a general antioxidant is shown in B. Data are given as means ± SEM. ^∗P < 0.05 versus Ec, ^†P < 0.05 versus control (con), and ^‡P < 0.05 versus DNP + Ec.

Figure 4

Metabolic stress–induced decreases in epithelial barrier function in human colon are inhibited by MitoTEMPO (MitoT). Biopsy specimens from normal human colon mounted in Ussing chambers and treated with 0.1 mmol/L DNP display a decrease in TER (A) and increased flux of ⁵¹Cr-EDTA (B), with the latter being significantly reduced by 10 μmol/L MitoT cotreatment (n = 6 to 11). C: DNP-induced increases in FITC-labeled dead E. coli (K12) flux is suppressed by MitoT (n = 6 to 11). One fluorescent unit equals 3.2 × 10³E. coli CFU (2-hour fluxes). Data are given as means ± SEM. ^∗P < 0.05 versus control (con) and DNP.

Figure 5

Tissues from patients with Crohn’s disease display increased baseline E. coli flux that is significantly enhanced by metabolic stress and reduced by MitoTEMPO. A: qPCR on biopsy specimens from patients with Crohn’s disease (each bar indicates a patient) reveals increased expression of peroxiredoxin-1 and ATP synthase mRNA, indicative of a response to metabolic stress. B:E. coli flux across biopsy specimens from patients with Crohn’s disease mounted in Ussing chambers is significantly increased by 0.1 mmol/L DNP, and this barrier defect is inhibited by 10 μmol/L MitoTEMPO (MitoT). One fluorescent unit equals 3.2 × 10³E. coli CFU (2-hour fluxes). ^∗P < 0.05, ^∗∗P < 0.01 versus the indicated group. Con, control.

Figure 6

Increased intestinal permeability is an early feature of DSS-induced colitis. BALB/c mice were exposed to 5% DSS for 3 days, with or without 100 μg MitoTEMPO (MitoT; i.p., daily), and colitis was assessed (Figure 1). A and B: DSS-treated mice appear normal, but on autopsy show signs of mild macroscopic disease (n = 11 to 12, three experiments) and histological damage (n = 3 to 4) (compare to Figure 7) that is reduced in Mito + DSS-treated mice. C: Lumen-to-blood flux of FITC-dextran (4 kDa) increases in the DSS-treated mice and is inhibited by MitoT (n = 7 to 8, two experiments). D: Immunoblotting of whole cell extracts of isolated colonic epithelial cells reveals a modest reduction in the mitochondria-specific VDAC protein. Data are given as means ± SEM. ^∗P < 0.05 versus control (cont), ^†P < 0.05 versus DSS. Original magnification, ×200 (H&E images). L, lumen of colon; m, external muscle layers.

Figure 7

A: MitoTEMPO (MitoT) suppresses colitis (n = 14) that is accompanied by enhanced barrier function measured by FITC-dextran flux (n = 3) and translocation of bacteria into the mucosa (n = 5 to 10). B–D: The anticolitic benefit of MitoTEMPO in the prolonged DSS model (Figure 1); MitoTEMPO used as either a prophylactic or a treatment significantly reduces macroscopic and histological disease. Data are given as means ± SEM [n = 15 to 19 mice from four experiments and five mice from one experiment in the prophylactic (III) and treatment (IV) regimens, respectively]. ^∗P < 0.05 versus control (con), ^†P < 0.05 versus DSS. Original magnification, ×200 (H&E images). L, lumen of colon; m, external muscle layers.

Figure 8

IL-8 production by T84 epithelia exposed to DSS is suppressed by cotreatment with 10 μmol/L Provided reagents MitoTEMPO (MitoT). Data are given as means ± SEM (n = 9 cell preparations from three separate experiments). ^∗P < 0.05 versus control, ^†P < 0.05 versus DSS.